DNA encoding a human melanin concentrating hormone receptor (MCH1) and uses thereof

ABSTRACT

This invention provides an isolated nucleic acid encoding a human MCH1 receptor, a purified human MCH1 receptor, vectors comprising isolated nucleic acid encoding a human MCH1 receptor, cells comprising such vectors, antibodies directed to a human MCH1 receptor, nucleic acid probes useful for detecting nucleic acid encoding human MCH1 receptors, antisense oligonucleotides complementary to unique sequences of nucleic acid encoding human MCH1 receptors, transgenic, nonhuman animals which express DNA encoding a normal or mutant human MCH1 receptor, methods of isolating a human MCH1 receptor, methods of treating an abnormality that is linked to the activity of a human MCH1 receptor, as well as methods of determining binding of compounds to mammalian MCH1 receptors.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced inparentheses by author and year. Full citations for these references maybe found at the end of the specification immediately preceding thesequence listings and the claims. The disclosure of these publicationsin their entireties are hereby incorporated by reference into thisapplication to describe more fully the state of the art to which thisinvention pertains.

Neuroregulators comprise a diverse group of natural products thatsubserve or modulate communication in the nervous system. They include,but are not limited to, neuropeptides, amino acids, biogenic amines,lipids and lipid metabolites, and other metabolic byproducts.

Many of these neuroregulator substances interact with specific cellsurface receptors which transduce signals from the outside to the insideof the cell. G-protein coupled receptors (GPCRs) represent a major classof cell surface receptors with which many neurotransmitters interact tomediate their effects.

GPCRs are predicted to have seven membrane-spanning domains and arecoupled to their effectors via G-proteins linking receptor activationwith intracellular biochemical sequelae such as stimulation of adenylylcyclase.

Melanin-concentrating hormone (MCH) is a cyclic peptide originallyisolated from salmonid (teleost fish) pituitaries (Kawauchi et al.,1983). In fish the 17 amino acid peptide causes aggregation of melaninwithin the melanophores and inhibits the release of ACTH, acting as afunctional antagonist of α-MSH. Mammalian MCH (19 amino acids) is highlyconserved between rat, mouse, and human, exhibiting 100% amino acididentity, but its physiological roles are less clear. MCH has beenreported to participate in a variety of processes including feeding,water balance, energy metabolism, general arousal/attention state,memory and cognitive functions, and psychiatric disorders (for reviews,see Baker, 1991; Baker, 1994; Nahon, 1994; Knigge et al., 1996). Itsrole in feeding or body weight regulation is supported by a recentNature publication (Qu et al., 1996) demonstrating that MCH isoverexpressed in the hypothalamus of ob/ob mice compared with ob/+ mice,and that fasting further increased MCH mRNA in both obese and normalmice during fasting. MCH also stimulated feeding in normal rats wheninjected into the lateral ventricles (Rossi et al., 1997). MCH also hasbeen reported to functionally antagonize the behavioral effects of α-MSH(Miller et al., 1993; Gonzalez et al, 1996; Sanchez et al., 1997); inaddition, stress has been shown to increase POMC mRNA levels whiledecreasing the MCH precursor preproMCH (ppMCH) mRNA levels (Presse etal., 1992). Thus MCH may serve as an integrative neuropeptide involvedin the reaction to stress, as well as in the regulation of feeding andsexual activity (Baker, 1991; Knigge et al., 1996).

The gene encoding the MCH precursor (ppMCH) has been cloned and encodestwo additional peptides, neuropeptide EI (13 AA) and neuropeptide GE(19AA) (Nahon et al., 1989), which may also have biological activity.MCH peptide is synthesized primarily in hypothalamic neurons (the zonaincerta and lateral hypothalamus) which project diffusely to many brainareas and to the pituitary (Bittencourt et al., 1992); NEI has also beenidentified in medium from explanted hypothalamic neurons (Parkes andVale, 1993). Localization studies of the mRNA indicate that MCH is alsopresent in the periphery (testes and GI tract; Hervieu and Nahon, 1995)but the highest concentrations are in the hypothalamus. There is alsoevidence for differential tissue-dependent processing of proMCH inmammals. A shorter MCH gene transcript that may result from alternatesplicing was found in several brain areas and peripheral tissues, and adifferent peptide form was also found in the periphery (Viale et al.,1997). In humans, the gene encoding authentic MCH has been localized tochromosome 12, but two copies of a variant (truncated) gene are presenton chromosome 5 (Breton et al., 1993); the functional significance, ifany, of the variant is not yet known. Finally, the rat MCH gene mayencode an additional putative peptide in a different reading frame(Toumaniantz et al., 1996).

Although the biological effects of MCH are believed to be mediated byspecific receptors, binding sites for MCH have not been well described.A tritiated ligand ([³H]-MCH) was reported to exhibit specific bindingto brain membranes but was unusable for saturation analyses, so neitheraffinity nor B_(max) were determined (Drozdz and Eberle, 1995).Radioiodination of the tyrosine at position thirteen resulted in aligand with dramatically reduced biological activity (see Drozdz andEberle, 1995). In contrast, the radioiodination of the MCH analogue[Phe¹³,Tyr¹⁹]-MCH was successful (Drozdz et al., 1995); the ligandretained biological activity and exhibited specific binding to a varietyof cell lines including mouse melanoma (B16-F1, G4F, and G4F-7), PC12,and COS cells. In G4F-7 cells, the K_(D)=0.118 nM and the B_(max) ˜1100sites/cell. Importantly, the binding was not inhibited by α-MSH but wasweakly inhibited by rat ANF (Ki=116 nM vs. 12 nM for native MCH) (Drozdzet al., 1995). More recently specific MCH binding was reported intransformed keratinocytes (Burgaud et al., 1997) and melanoma cells(Drozdz et al., 1998), where photo-crosslinking studies suggest that thereceptor is a membrane protein with an apparent molecular weight of45-50 kDaltons, compatible with the molecular weight range of the GPCRsuperfamily of receptors. No radioautoradiographic studies of MCHreceptor localization using this ligand have been reported as yet.

Signal transduction mechanisms for MCH receptors remain obscure. Nodirect evidence supporting G-protein coupling exists in mammals, but twolines of weak evidence exist in teleost fish for G_(αq)- and/orG_(60 i)-type coupling: 1) indirect evidence exists for MCH acting viaphospholipase C in teleost fish melanophores (phospholipase C inhibitorsand protein kinase C inhibitors shift the MCH dose-response curve to theright, and TPA mimics MCH at low doses (Abrao et al., 1991)); and 2)MCH-elicited pigment aggregation in fish melanophores is associated witha reduction in basal cAMP levels, similar to that observed withnorepinephrine (Svensson et al., 1991; Morishita et al., 1993). Arguingagainst G-protein coupling is the general structural homology of MCHwith ANF, whose receptors are not in the GPCR superfamily. Recently theactions of MCH were reported to be mediated via activation of aphosphatidylinositol-3-kinase pathway which is typical of tyrosinekinase and cytokine receptors (Qu et al., 1998); however, since multiplesignaling pathways (receptor cross talk) may produce this mediator noconclusions can be reached regarding MCH signal transduction pathways inmammalian systems.

The localization and biological activities of MCH peptide suggest thatthe modulation of MCH receptor activity may be useful in a number oftherapeutic applications. The role of MCH in feeding is the bestcharacterized of its potential clinical uses. MCH is expressed in thelateral hypothalamus, a brain area implicated in the regulation ofthirst and hunger (Grillon et al., 1997); recently orexins A and B,which are potent orexigenic agents, have been shown to have very similarlocalization to MCH in the lateral hypothalamus (Sakurai et al., 1998).MCH mRNA levels in this brain region are increased in rats after 24hours of food-deprivation (Herve and Fellman, 1997); after insulininjection, a significant increase in the abundance and stainingintensity of MCH immunoreactive perikarya and fibres was observedconcurrent with a significant increase in the level of MCH mRNA(Bahjaoui-Bouhaddi et al., 1994). Consistent with the ability of MCH tostimulate feeding in rats (Rossi et al., 1997) is the observation thatMCH mRNA levels are upregulated in the hypothalami of obese ob/ob mice(Qu et al., 1996), and decreased in the hypothalami of rats treated withleptin, whose food intake and body weight gains are also decreased(Sahu, 1998). MCH appears to act as a functional antagonist of themelanocortin system in its effects on food intake and on hormonesecretion within the HPA (hypothalamopituitary/adrenal axis) (Ludwig etal., 1998). Together these data suggest a role for endogenous MCH in theregulation of energy balance and response to stress, and provide arationale for the development of specific compounds acting at MCHreceptors for use in the treatment of obesity and stress-relateddisorders.

In all species studied to date, a major portion of the neurons of theMCH cell group occupies a rather constant location in those areas of thelateral hypothalamus and subthalamus where they lie and may be a part ofsome of the so-called “extrapyramidal” motor circuits. These involvesubstantial striato- and pallidofugal pathways involving the thalamusand cerebral cortex, hypothalamic areas, and reciprocal connections tosubthalamic nucleus, substantia nigra, and mid-brain centers(Bittencourt et al., 1992). In their location, the MCH cell group mayoffer a bridge or mechanism for expressing hypothalamic visceralactivity with appropriate and coordinated motor activity. Clinically itmay be of some value to consider the involvement of this MCH system inmovement disorders, such as Parkinson's disease and Huntingdon's Choreain which extrapyramidal circuits are known to be involved.

Human genetic linkage studies have located authentic hMCH loci onchromosome 12 (12q23-24) and the variant hMCH loci on chromosome 5(5q12-13) (Pedeutour et al., 1994). Locus 12q23-24 coincides with alocus to which autosomal dominant cerebellar ataxia type II (SCA2) hasbeen mapped (Auburger et al., 1992; Twells et al., 1992). This diseasecomprises neurodegenerative disorders, including an olivopontocerebellaratrophy.

Furthermore, the gene for Darier's disease, has been mapped to locus12q23-24 (Craddock et al., 1993). Dariers' disease is characterized byabnormalities I keratinocyte adhesion and mental illnesses in somefamilies. In view of the functional and neuroanatomical patterns of theMCH neural system in the rat and human brains, the MCH gene mayrepresent a good candidate for SCA2 or Darier's disease. Interestingly,diseases with high social impact have been mapped to this locus. Indeed,the gene responsible for chronic or acute forms of spinal muscularatrophies has been assigned to chromosome 5q12-13 using genetic linkageanalysis (Melki et al., 1990; Westbrook et al., 1992). Furthermore,independent lines of evidence support the assignment of a majorschizophrenia locus to chromosome 5q11.2-13.3 (Sherrington et al., 1988;Bassett et al., 1988; Gilliam et al., 1989). The above studies suggestthat MCH may play a role in neurodegenerative diseases and disorders ofemotion.

Additional therapeutic applications for MCH-related compounds aresuggested by the observed effects of MCH in other biological systems.For example, MCH may regulate reproductive functions in male and femalerats. MCH transcripts and MCH peptide were found within germ cells intestes of adult rats, suggesting that MCH may participate in stem cellrenewal and/or differentiation of early spermatocytes (Hervieu et al.,1996). MCH injected directly into the medial preoptic area (MPOA) orventromedial nucleus (VMN) stimulated sexual activity in female rats(Gonzalez et al., 1996). In ovariectomized rats primed with estradiol,MCH stimulated luteinizing hormone (LH) release while anti-MCH antiseruminhibited LH release (Gonzalez et al., 1997). The zona incerta, whichcontains a large population of MCH cell bodies, has previously beenidentified as a regulatory site for the pre-ovulatory LH surge(MacKenzie et al., 1984). MCH has been reported to influence release ofpituitary hormones including ACTH and oxytocin. MCH analogues may alsobe useful in treating epilepsy. In the PTZ seizure model, injection ofMCH prior to seizure induction prevented seizure activity in both ratsand guinea pigs, suggesting that MCH-containing neurons may participatein the neural circuitry underlying PTZ-induced seizure (Knigge andWagner, 1997). MCH has also been observed to affect behavioralcorrelates of cognitive functions. MCH treatment hastened extinction ofthe passive avoidance response in rats (McBride et al., 1994), raisingthe possibility that MCH receptor antagonists may be beneficial formemory storage and/or retention. A possible role for MCH in themodulation or perception of pain is supported by the dense innervationof the periaqueductal grey (PAG) by MCH-positive fibers. Finally, MCHmay participate in the regulation of fluid intake. ICV infusion of MCHin conscious sheep produced diuretic, natriuretic, and kaliureticchanges in response to increased plasma volume (Parkes, 1996). Togetherwith anatomical data reporting the presence of MCH in fluid regulatoryareas of the brain, the results indicate that MCH may be an importantpeptide involved in the central control of fluid homeostasis in mammals.

SUMMARY OF THE INVENTION

This invention provides an isolated nucleic acid encoding a human MCH1receptor.

This invention provides a nucleic acid encoding a human MCH1 receptor,wherein the nucleic acid (a) hybridizes to a nucleic acid having thedefined sequence shown in FIG. 1 (Seq. ID No. 1) under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when an MCH1 ligand is added to the culture and the CHO cellscontain the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement.

This invention provides a purified human MCH1 receptor protein.

This invention provides a vector comprising a nucleic acid encoding ahuman MCH1 receptor, particularly a vector adapted for expression of thehuman MCH1 receptor in mammalian or non-mammalian cells. One such vectoris a plasmid designated pEXJ.HR-TL231 (ATCC Accession No. 203197) whichcomprises a nucleotide sequence encoding a human MCH1 receptor.

This invention also provides a cell comprising a vector which comprisesa nucleic acid encoding a human MCH1 receptor as well as a membranepreparation isolated from such cells.

This invention further provides a nucleic acid probe comprising at least15 nucleotides which specifically hybridizes with a nucleic acidencoding a mammalian MCH1 receptor, wherein the probe has a uniquesequence corresponding to a sequence present within the nucleic acidwhich encodes the human MCH1 receptor or its complement, both of whichare present in plasmid pEXJ.HR-TL231 (ATCC Accession No. 203197).

This invention further provides a nucleic acid probe comprising at least15 nucleotides which specifically hybridizes with a nucleic acidencoding a mammalian MCH1 receptor, wherein the probe has a uniquesequence corresponding to a sequence present within (a) the nucleic acidsequence shown in FIG. 1 (Seq. I.D. No. 1) or (b) the reverse complementthereof.

This invention also provides an antisense oligonucleotide having asequence capable of specifically hybridizing an RNA encoding a humanMCH1 receptor, so as to prevent translation of the RNA and an antisenseoligonucleotide having a sequence capable of specifically hybridizing tothe genomic DNA encoding a human MCH1 receptor.

This invention further provides an antibody capable of binding to ahuman MCH1 receptor as well as an agent capable of competitivelyinhibiting the binding of the antibody to a human MCH1 receptor.

This invention provides a pharmaceutical composition comprising (a) anamount of the oligonucleotide described above capable of passing througha cell membrane and effective to reduce expression of a human MCH1receptor and (b) a pharmaceutically acceptable carrier capable ofpassing through the cell membrane.

Moreover, this invention provides a transgenic, nonhuman mammalexpressing DNA encoding a human MCH1 receptor. This invention alsoprovides a transgenic, nonhuman mammal comprising a homologousrecombination knockout of the native human MCH1 receptor. This inventionfurther provides a transgenic, nonhuman mammal whose genome comprisesantisense DNA complementary to the DNA encoding a human MCH1 receptor soplaced within the genome as to be transcribed into antisense mRNA whichis complementary to mRNA encoding the human MCH1 receptor and whichhybridizes to mRNA encoding the human MCH1 receptor, thereby reducingits translation.

In one embodiment this invention provides a process for identifying achemical compound which specifically binds to a mammalian MCH1 receptorwhich comprises contacting cells containing DNA encoding and expressingon their cell surface a mammalian MCH1 receptor, wherein such cells donot normally express the mammalian MCH1 receptor, with the compoundunder conditions suitable for binding, and detecting specific binding ofthe chemical compound to the mammalian MCH1 receptor.

This invention provides a process for identifying a chemical compoundwhich specifically binds to a mammalian MCH1 receptor which comprisescontacting a membrane preparation from cells transfected with DNAencoding and expressing on their cell surface the mammalian MCH1receptor, wherein such cells do not normally express the mammalian MCH1receptor, with the compound under conditions suitable for binding, anddetecting specific binding of the chemical compound to the mammalianMCH1 receptor.

This invention provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a mammalianMCH1 receptor which comprises separately contacting cells expressing ontheir cell surface the mammalian MCH1 receptor, wherein such cells donot normally express the mammalian MCH1 receptor, with both the chemicalcompound and a second chemical compound known to bind to the receptor,and with only the second chemical compound, under conditions suitablefor binding of both compounds, and detecting specific binding of thechemical compound to the mammalian MCH1 receptor, a decrease in thebinding of the second chemical compound to the mammalian MCH1 receptorin the presence o f the chemical compound indicating that the chemicalcompound binds to the mammalian MCH1 receptor.

This invention provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a mammalianMCH1 receptor which comprises separately contacting a membrane fractionfrom a cell extract of cells expressing on their cell surface themammalian MCH1 receptor, wherein such cells do not normally express themammalian MCH1 receptor, with both the chemical compound and a secondchemical compound known to bind to the receptor, and with only thesecond chemical compound, under conditions suitable for binding of bothcompounds, and detecting specific binding of the chemical compound tothe mammalian MCH1 receptor, a decrease in the binding of the secondchemical compound to the mammalian MCH1 receptor in the presence of thechemical compound indicating that the chemical compound binds to themammalian MCH1 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian MCH1 receptor to identify acompound which specifically binds to the mammalian MCH1 receptor, whichcomprises (a) contacting cells transfected with and expressing DNAencoding the mammalian MCH1 receptor with a compound known to bindspecifically to the mammalian MCH1 receptor; (b) contacting thepreparation of step (a) with the plurality of compounds not known tobind specifically to the mammalian MCH1 receptor, under conditionspermitting binding of compounds known to bind the mammalian MCH1receptor; (c) determining whether the binding of the compound known tobind to the mammalian MCH1 receptor is reduced in the presence of thecompounds within the plurality of compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (d)separately determining the binding to the mammalian MCH1 receptor ofcompounds included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the mammalian MCH1receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian MCH1 receptor to identify acompound which specifically binds to the mammalian MCH1 receptor, whichcomprises (a) contacting a membrane preparation from cells transfectedwith and expressing DNA encoding a mammalian MCH1 receptor with acompound known to bind specifically to the mammalian MCH1 receptor; (b)contacting the preparation of step (a) with the plurality of compoundsnot known to bind specifically to the mammalian MCH1 receptor, underconditions permitting binding of compounds known to bind the mammalianMCH1 receptor; (c) determining whether the binding of the compound knownto bind to the mammalian MCH1 receptor is reduced in the presence of thecompounds within the plurality of compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (d)separately determining the binding to the mammalian MCH1 receptor ofcompounds included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the mammalian MCHIreceptor.

This invention provides a method of detecting expression of a mammalianMCH1 receptor by detecting the presence of mRNA coding for the mammalianMCH1 receptor which comprises obtaining total mRNA from the cell andcontacting the mRNA so obtained with a nucleic acid probe underhybridizing conditions, detecting the presence of mRNA hybridizing tothe probe, and thereby detecting the expression of the mammalian MCH1receptor by the cell.

This invention provides a method of detecting the presence of amammalian MCH1 receptor on the surface of a cell which comprisescontacting the cell with an antibody under conditions permitting bindingof the antibody to the receptor, detecting the presence of the antibodybound to the cell, and thereby detecting the presence of the mammalianMCH1 receptor on the surface of the cell.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of human MCH1 receptors whichcomprises producing a transgenic, nonhuman mammal whose levels of humanMCH1 receptor activity are varied by use of an inducible promoter whichregulates human MCH1 receptor expression.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of human MCH1 receptors whichcomprises producing a panel of transgenic, nonhuman mammals eachexpressing a different amount of human MCH1 receptor.

This invention provides a method for identifying an antagonist capableof alleviating an abnormality wherein the abnormality is alleviated bydecreasing the activity of a human MCH1 receptor comprisingadministering a compound to the transgenic, nonhuman mammal anddetermining whether the compound alleviates the physical and behavioralabnormalities displayed by the transgenic, nonhuman mammal as a resultof overactivity of a human MCH1 receptor, the alleviation of theabnormality identifying the compound as an antagonist. This inventionalso provides an antagonist identified by this method. This inventionfurther provides a pharmaceutical composition comprising an antagonistidentified by this method and a pharmaceutically acceptable carrier.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by decreasing the activity of ahuman MCH1 receptor which comprises administering to the subject aneffective amount of this pharmaceutical composition, thereby treatingthe abnormality.

This invention provides a method for identifying an agonist capable ofalleviating an abnormality in a subject wherein the abnormality isalleviated by increasing the activity of a human MCH1 receptorcomprising administering a compound to a transgenic, nonhuman mammal,and determining whether the compound alleviates the physical andbehavioral abnormalities displayed by the transgenic, nonhuman mammal,the alleviation of the abnormality identifying the compound as anagonist. This invention also provides an agonist identified by thismethod. This invention further provides a pharmaceutical compositioncomprising an agonist identified by this method and a pharmaceuticallyacceptable carrier. This invention provides a method of treating anabnormality in a subject wherein the abnormality is alleviated byincreasing the activity of a human MCH1 receptor which comprisesadministering to the subject an effective amount of this pharmaceuticalcomposition, thereby treating the abnormality.

This invention provides a method for diagnosing a predisposition to adisorder associated with the activity of a specific mammalian allelewhich comprises: (a) obtaining DNA of subjects suffering from thedisorder; (b) performing a restriction digest of t he DNA with a panelof restriction enzymes; (c) electrophoretically separating the resultingDNA fragments on a sizing gel; (d) contacting the resulting gel with anucleic acid probe capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a human MCH1 receptor and labeled with a detectable marker; (e)detecting labeled bands which have hybridized to thee DNA encoding ahuman MCH1 receptor labeled with a detectable marker to create a uniqueband pattern specific to the DNA of subjects suffering from thedisorder; (f) preparing DNA obtained for diagnosis by steps (a)-(e) ;and (g) comparing the unique band pattern specific to the DNA ofsubjects suffering from the disorder from step (e) and the DNA obtainedfor diagnosis from step (f) to determine whether the patterns are thesame or different and to diagnose thereby predisposition to the disorderif the patterns are the same.

This invention provides a method of preparing a purified human MCH1receptor which comprises: (a) inducing cells to express the human MCH1receptor; (b) recovering the human MCH1 receptor from the induced cells;and (c) purifying the human MCH1 receptor so recovered.

This invention provides a method of preparing a purified human MCH1receptor which comprises: (a) inserting nucleic acid encoding the humanMCH1 receptor in a suitable vector; (b) introducing the resulting vectorin a suitable host cell; (c) placing the resulting cell in suitablecondition permitting the production of the isolated human MCH1 receptor;(d) recovering the human MCH1 receptor produced by the resulting cell;and (e) purifying the human MCH1 receptor so recovered.

This invention provides a process for determining whether a chemicalcompound is a mammalian MCH1 receptor agonist which comprises contactingcells transfected with and expressing DNA encoding the mammalian MCH1receptor with the compound under conditions permitting the activation ofthe mammalian MCH1 receptor, and detecting an increase in mammalian MCH1receptor activity, so as to thereby determine whether the compound is amammalian MCH1 receptor agonist. This invention also provides apharmaceutical composition which comprises an amount of a mammalian MCH1receptor agonist determined by this process effective to increaseactivity of a mammalian MCH1 receptor and a pharmaceutically acceptablecarrier.

This invention provides a process for determining whether a chemicalcompound is a mammalian MCH1 receptor antagonist which comprisescontacting cells transfected with and expressing DNA encoding themammalian MCH1 receptor with the compound in the presence of a knownmammalian MCH1 receptor agonist, under conditions permitting theactivation of the mammalian MCH1 receptor, and detecting a decrease inmammalian MCH1 receptor activity, so as to thereby determine whether thecompound is a mammalian MCH1 receptor antagonist. This invention alsoprovides a pharmaceutical composition which comprises an amount of amammalian MCH1 receptor antagonist determined by this process effectiveto reduce activity of a mammalian MCH1 receptor and a pharmaceuticallyacceptable carrier.

This invention provides a process for determining whether a chemicalcompound specifically binds to and activates a mammalian MCH1 receptor,which comprises contacting cells producing a second messenger responseand expressing on their cell surface the mammalian MCH1 receptor,wherein such cells do not normally express the mammalian MCH1 receptor,with the chemical compound under conditions suitable for activation ofthe mammalian MCH1 receptor, and measuring the second messenger responsein the presence and in the absence of the chemical compound, a change inthe second messenger response in the presence of the chemical compoundindicating that the compound activates the mammalian MCH1 receptor. Thisinvention also provides a compound determined by this process. Thisinvention further provides a pharmaceutical composition which comprisesan amount of the compound (a MCH1 receptor agonist) determined by thisprocess effective to increase activity of a mammalian MCH1 receptor anda pharmaceutically acceptable carrier.

This invention provides a process for determining whether a chemicalcompound specifically binds to and inhibits activation of a mammalianMCH1 receptor, which comprises separately contacting cells producing asecond messenger response and expressing on their cell surface themammalian MCH1 receptor, wherein such cells do not normally express themammalian MCH1 receptor, with both the chemical compound and a secondchemical compound known to activate the mammalian MCH1 receptor, andwith only the second chemical compound, under conditions suitable foractivation of the mammalian MCH1 receptor, and measuring the secondmessenger response in the presence of only the second chemical compoundand in the presence of both the second chemical compound and thechemical compound, a smaller change in the second messenger response inthe presence of both the chemical compound and the second chemicalcompound than in the presence of only the second chemical compoundindicating that the chemical compound inhibits activation of themammalian MCH1 receptor. This invention also provides a compounddetermined by this process. This invention further provides apharmaceutical composition which comprises an amount of the compound (amammalian MCH1 receptor antagonist) determined by this effective toreduce activity of a mammalian MCH1 receptor and a pharmaceuticallyacceptable carrier.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a mammalian MCH1 receptor to identify acompound which activates the mammalian MCH1 receptor which comprises:(a) contacting cells transfected with and expressing the mammalian MCH1receptor with the plurality of compounds not known to activate themammalian MCH1 receptor, under conditions permitting activation of themammalian MCH1 receptor; (b) determining whether the activity of themammalian MCH1 receptor is increased in the presence of the compounds;and if so (c) separately determining whether the activation of themammalian MCH1 receptor is increased by each compound included in theplurality of compounds, so as to thereby identify the compound whichactivates the mammalian MCH1 receptor. This invention also provides acompound identified by this method. This invention further provides apharmaceutical composition which comprises an amount of the compound (amammalian MCH1 receptor agonist) identified by this method effective toincrease activity of a mammalian MCH1 receptor and a pharmaceuticallyacceptable carrier.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a mammalian MCH1receptor to identify a compound which inhibits the activation of themammalian MCH1 receptor, which comprises: (a) contacting cellstransfected with and expressing the mammalian MCH1 receptor with theplurality of compounds in the presence of a known mammalian MCH1receptor agonist, under conditions permitting activation of themammalian MCH1 receptor; (b) determining whether the activation of themammalian MCH1 receptor is reduced in the presence of the plurality ofcompounds, relative to the activation of the mammalian MCH1 receptor inthe absence of the plurality of compounds; and if so (c) separatelydetermining the inhibition of activation of the mammalian MCH1 receptorfor each compound included in the plurality of compounds, so as tothereby identify the compound which inhibits the activation of themammalian MCH1 receptor. This invention also provides a compoundidentified by this method. This invention further provides apharmaceutical composition which comprises an amount of the compound (amammalian MCH1 receptor antagonist) identified by this process effectiveto decrease activity of a mammalian MCH1 receptor and a pharmaceuticallyacceptable carrier.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by increasing the activity of amammalian MCH1 receptor which comprises administering to the subject anamount of a compound which is a mammalian MCH1 receptor agonisteffective to treat the abnormality.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by decreasing the activity of amammalian MCH1 receptor which comprises administering to the subject anamount of a compound which is a mammalian MCH1 receptor antagonisteffective to treat the abnormality.

This invention provides a process for making a composition of matterwhich specifically binds to a mammalian MCH1 receptor which comprisesidentifying a chemical compound using any of the processes describedherein for identifying a compound which binds to and/or activates orinhibits activation of a mammalian MCH1 receptor and then synthesizingthe chemical compound or a novel structural and functional analog orhomolog thereof. This invention further provides a process for preparinga pharmaceutical composition which comprises administering apharmaceutically acceptable carrier and a pharmaceutically acceptableamount of a chemical compound identified by any of the processesdescribed herein for identifying a compound which binds to and/oractivates or inhibits activation of a mammalian MCH1 receptor or a novelstructural and functional analog or homolog thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Nucleotide sequence encoding a human MCH1 receptor (MCH1) (Seq.I.D. No. 1). Three potential start (ATG) codons and the stop (TGA) codonare underlined.

FIG. 2 Deduced amino acid sequence (Seq. I.D. No. 2) of the human MCH1receptor (MCH1) encoded by the nucleotide sequence shown FIG. 1 (Seq.I.D. No. 1).

FIG. 3 Deduced amino acid sequence for human MCH1 (SEQ. ID No. 2). Theseven putative transmembrane (TM) regions are underlined.

FIG. 4 Nucleotide sequence of rat MCH1 (SEQ. ID No. 3). One start (ATG)codon and the stop codon (TGA) are underlined.

FIG. 5 Deduced amino acid sequence for rat MCH1 (SEQ. ID No. 4).

FIG. 6 MCH1-mediated IP dose response to MCH.

FIG. 7 MCH1 challenge with several compounds of interest.

FIG. 8 MCH1-mediated extracellular acidification response to MCH andPhe¹³,Tyr¹⁹-MCH. Results are reported as the average of two independentexperiments performed in duplicate.

FIG. 9 Transcriptional response of MCH1-transfected Cos-7 cells to MCH.

FIG. 10 Binding of [¹²⁵I]Phe¹³,Tyr¹⁹-MCH on MCH1-transfected Cos-7 cellmembranes. Results are means±S.E.M. (vertical lines) of triplicatedeterminations.

FIG. 11 RT-PCR detection of MCH1 receptor mRNA in human mRNA samples.

DETAILED DESCRIPTION OF THE INVENTION

Throughout this application, the following standard abbreviations areused to indicate specific nucleotide bases:

A=adenine

G=guanine

C=cytosine

T=thymine

U=uracil

M=adenine or cytosine

R=adenine or guanine

W=adenine, thymine, or uracil

S=cytosine or guanine

Y=cytosine, thymine, or uracil

K=guanine, thymine, or uracil

V=adenine, cytosine, or guanine (not thymine or uracil

H=adenine, cytosine, thymine, or uracil (not guanine)

D=adenine, guanine, thymine, or uracil (not cytosine)

B=cytosine, guanine, thymine, or uracil (not adenine)

N=adenine, cytosine, guanine, thymine, or uracil (or other modified basesuch as inosine)

I=inosine

Furthermore, the term “agonist” is used throughout this application toindicate any peptide or non-peptidyl compound which increases theactivity of any of the polypeptides of the subject invention. The term“antagonist” is used throughout this application to indicate any peptideor non-peptidyl compound which decreases the activity of any of thepolypeptides of the subject invention.

The activity of a G-protein coupled receptor such as the polypeptidesdisclosed herein may be measured using any of a variety of functionalassays in which activation of the receptor in question results in anobservable change in the level of some second messenger system,including, but not limited to, adenylate cyclase, calcium mobilization,arachidonic acid release, ion channel activity, inositol phospholipidhydrolysis or guanylyl cyclase. Heterologous expression systemsutilizing appropriate host cells to express the nucleic acid of thesubject invention are used to obtain the desired second messengercoupling. Receptor activity may also be assayed in an oocyte expressionsystem.

It is possible that the human MCH1 receptor gene contains introns andfurthermore, the possibility exists that additional introns could existin coding or non-coding regions. In addition, spliced form(s) of mRNAmay encode additional amino acids either upstream of the currentlydefined starting methionine or within the coding region. Further, theexistence and use of alternative exons is possible, whereby the mRNA mayencode different amino acids within the region comprising the exon. Inaddition, single amino acid substitutions may arise via the mechanism ofRNA editing such that the amino acid sequence of the expressed proteinis different than that encoded by the original gene. (Burns et al.,1996; Chu et al., 1996).

Such variants may exhibit pharmacologic properties differing from thepolypeptide encoded by the original gene.

This invention provides splice variants of the human MCH1 receptordisclosed herein. This invention further provides for alternatetranslation initiation sites and alternately spliced or edited variantsof nucleic acids encoding the human MCH1 receptor of this invention.

The nucleic acid of the subject invention also includes nucleic acidanalogs of the human MCH1 receptor gene, wherein the human MCH1 receptorgene comprises the nucleic acid sequence shown in FIG. 1 or contained inplasmid pEXJ.HR-TL231 (ATCC Accession No. 203197). Nucleic acid analogsof the human MCH1 receptor genes differ from the human MCH1 receptorgene described herein in terms of the identity or location of one ormore nucleic acid bases (deletion analogs containing less than all ofthe nucleic acid bases shown in FIG. 1 or contained in plasmidpEXJ.HR-TL231, substitution analogs wherein one or more nucleic acidbases shown in FIG. 1 or contained in plasmids pEXJ.HR-TL231 arereplaced by other nucleic acid bases, and addition analogs, wherein oneor more nucleic acid bases are added to a terminal or medial portion ofthe nucleic acid sequence) and which encode proteins which share some orall of the properties of the proteins encoded by the nucleic acidsequences shown in FIG. 1 or contained in plasmid pEXJ.HR-TL231. In oneembodiment of the present invention, the nucleic acid analog encodes aprotein which comprises an amino acid sequence as shown in FIG. 2 orencoded by the nucleic acid sequence contained in plasmid pEXJ.HR-TL231.In another embodiment, the nucleic acid analog encodes a proteincomprising an amino acid sequence which differs from the amino acidsequences shown in FIG. 2 or encoded by the nucleic acid contained inplasmids pEXJ.HR-TL231. In a further embodiment, the protein encoded bythe nucleic acid analog has a function which is the same as the functionof the receptor protein comprising the amino acid sequence shown in FIG.2. In another embodiment, the function of the protein encoded by thenucleic acid analog differs from the function of the receptor proteincomprising the amino acid sequence shown in FIG. 2. In anotherembodiment, the variation in the nucleic acid sequence occurs within thetransmembrane (TM) region of the protein. In a further embodiment, thevariation in the nucleic acid sequence occurs outside of the TM region.

This invention provides the above-described isolated nucleic acid,wherein the nucleic acid is DNA. In an embodiment, the DNA is cDNA. Inanother embodiment, the DNA is genomic DNA. In still another embodiment,the nucleic acid is RNA. Methods for production and manipulation ofnucleic acid molecules are well known in the art.

This invention further provides nucleic acid which is degenerate withrespect to the DNA encoding the polypeptides described herein. In anembodiment, the nucleic acid comprises a nucleotide sequence which isdegenerate with respect to the nucleotides sequence shown in FIG. 1 (SEQID NO. 2) or the nucleotide sequence contained in the plasmidpEXJ.HR-TL231, that is, a nucleotide sequence which is translated intothe same amino acid sequence.

This invention also encompasses DNAs and cDNAs which encode amino acidsequences which differ from those of the polypeptides of this invention,but which should not produce phenotypic changes. Alternately, thisinvention also encompasses DNAs, cDNAs, and RNAs which hybridize to theDNA, cDNA, and RNA of the subject invention. Hybridization methods arewell known to those of skill in the art.

The nucleic acids of the subject invention also include nucleic acidmolecules coding for polypeptide analogs, fragments or derivatives ofantigenic polypeptides which differ from naturally-occurring forms interms of the identity or location of one or more amino acid residues(deletion analogs containing less than all of the residues specified forthe protein, substitution analogs wherein one or more residues specifiedare replaced by other residues and addition analogs wherein one or moreamino acid residues is added to a terminal or medial portion of thepolypeptides) and which share some or all properties ofnaturally-occurring forms. These molecules include: the incorporation ofcodons “preferred” for expression by selected non-mammalian hosts; theprovision of sites for cleavage by restriction endonuclease enzymes; andthe provision of additional initial, terminal or intermediate DNAsequences that facilitate construction of readily expressed vectors. Thecreation of polypeptide analogs is well known to those of skill in theart (R. F. Spurney et al. (1997); Fong, T. M. et al. (1995); Underwood,D. J. et al. (1994); Graziano, M. P. et al. (1996); Guan X. M. et al.(1995)).

The modified polypeptides of this invention may be transfected intocells either transiently or stably using methods well-known in the art,examples of which are disclosed herein. This invention also provides forbinding assays using the modified polypeptides, in which the polypeptideis expressed either transiently or in stable cell lines. This inventionfurther provides a compound identified using a modified polypeptide in abinding assay such as the binding assays described herein.

The nucleic acids described and claimed herein are useful for theinformation which they provide concerning the amino acid sequence of thepolypeptide and as products for the large scale synthesis of thepolypeptides by a variety of recombinant techniques. The nucleic acidmolecule is useful for generating new cloning and expression vectors,transformed and transfected prokaryotic and eukaryotic host cells, andnew and useful methods for cultured growth of such host cells capable ofexpression of the polypeptide and related products.

This invention provides an isolated nucleic acid encoding a human MCH1receptor. In one embodiment, the nucleic acid is DNA. In anotherembodiment, the DNA is cDNA. In another embodiment, the DNA is genomicDNA. In another embodiment, the nucleic acid is RNA.

This invention also provides methods of using an isolated nucleic acidencoding species homologs of the MCH1 receptor encoded by the nucleicacid sequence shown in FIG. 1 (Seq. ID No. 1) or encoded by the plasmidpEXJ.HR-TL231. In one embodiment, the nucleic acid encodes a mammalianMCH1 receptor homolog which has substantially the same amino acidsequence as does the MCH1 receptor encoded by the plasmid pEXJ.HR-TL231.In another embodiment, the nucleic acid encodes a mammalian MCH1receptor Homolog which has above 65% amino acid identity to the MCH1receptor encoded by the plasmid pEXJ.HR-TL231; preferably above 75%amino acid identity to the MCH1 receptor encoded by the plasmidpEXJ.HR-TL231; more preferably above 85 amino acid identity to the MCH1receptor encoded by the plasmid pEXJ.HR-TL231; most preferably above 95%amino acid identity to the MCH1 receptor encoded by the plasmidpEXJ.HR-TL231. In another embodiment, the mammalian MCH1 receptorhomolog has above 70% nucleic acid identity to the MCH1 receptor genecontained in plasmid pEXJ.HR-TL231; preferably above 80% nucleic acididentity to the MCH1 receptor gene contained in the plasmidpEXJ.HR-TL231; more preferably above 90% nucleic acid identity to theMCH1 receptor gene contained in the plasmid pEXJ.HR-TL231. Examples ofmethods for isolating and purifying species homologs are describedelsewhere (e.g., U.S. Pat. No. 5,602,024, WO94/14957, WO97/26853,WO98/15570).

In a separate embodiment of the present invention, the nucleic acidencodes a MCH1 receptor which has an amino acid sequence identical tothat encoded by the plasmid pEXJ.HR-TL231. In a further embodiment, theMCH1 receptor comprises a sequence substantially the same as the aminoacid sequence shown in FIG. 2 (Seq. I.D. No. 2). In another embodiment,the MCH1 receptor comprises an amino acid sequence as shown in FIG. 2(Seq. I.D. No. 2).

In separate embodiments, the human MCH1 receptor is encoded by thenucleic acid sequence shown in FIG. 1 beginning with any of the threeindicated start (ATG) codons.

This invention provides an isolated nucleic acid encoding a modifiedhuman MCH1 receptor, which differs from a human MCH1 receptor by havingan amino acid(s) deletion, replacement, or addition in the thirdintracellular domain.

This invention provides a nucleic acid encoding a human MCH1 receptor,wherein the nucleic acid (a) hybridizes to a nucleic acid having thedefined sequence shown in FIG. 1 (Seq. ID No. 1) under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a MCH1 ligand is added to the culture and the CHO cellscontain the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement. Hybridization at low stringency isperformed at 40° C. in a hybridization buffer containing 25% formamide,5×SCC, 7 mM Tris, 1×Denhardt's, 25 μl/ml salmon sperm DNA. Wash at 40°C. in 0.1×SCC, 0.1 SDS. Changes in pH are measured throughmicrophysiometric measurement of receptor mediated extracellularacidification rates. Because cellular metabolism is intricately involvedin a broad range of cellular events (including receptor activation ofmultiple messenger pathways), the use of microphysiometric measurementsof cell metabolism can in principle provide a generic assay of cellularactivity arising from the activation of any receptor regardless of thespecifics of the receptor's signaling pathway. General guidelines fortransient receptor expression, cell preparation and microphysiometricrecording are described elsewhere (Salon, J. A. and Owicki, J. A.,1996). Receptors and/or control vectors are transiently expressed inCHO-K1 cells, by liposome mediated transfection according to themanufacturers recommendations (LipofectAMINE, GibcoBRL, Gaithersburg,Md.), and maintained in Ham's F-12 complete (10% serum). A total of 10μg of DNA is used to transfect each 75 cm² flask which had been split 24hours prior to the transfection and judged to be 70-80% confluent at thetime of transfection. 24 hours post transfection, the cells areharvested and 3×10⁵ cells seeded into microphysiometer capsules. Cellsare allowed to attach to the capsule membrane for an additional 24hours; during the last 16 hours, the cells are switched to serum-freeF-12 complete to minimize ill-defined metabolic stimulation caused byassorted serum factors. On the day of the experiment the cell capsulesare transferred to the microphysiometer and allowed to equilibrate inrecording media (low buffer RPMI 1640, no bicarbonate, no serum(Molecular Devices Corporation, Sunnyvale, Calif.) containing 0.1% fattyacid free BSA), during which a baseline measurement of basal metabolicactivity is established. A standard recording protocol specifies a 100μl/min flow rate, with a 2 min total pump cycle which includes a 30 secflow interruption during which the acidification rate measurement istaken. Ligand challenges involve a 1 min 20 sec exposure to the samplejust prior to the first post challenge rate measurement being taken,followed by two additional pump cycles for a total of 5 min 20 secsample exposure. Typically, drugs in a primary screen are presented tothe cells at 10 μM final concentration. Ligand samples are then washedout and the acidification rates reported are expressed as a percentageincrease of the peak response over the baseline rate observed just priorto challenge. An examples of a MCH ligand includes, but is not limitedto, the endogenous MCH peptide.

This invention provides a purified human MCH1 receptor protein.

This invention provides a vector comprising nucleic acid encoding ahuman MCH1 receptor. In an embodiment, the vector is adapted forexpression in a cell which comprises the regulatory elements necessaryfor expression of the nucleic acid in the cell operatively linked to thenucleic acid encoding the human MCH1 receptor as to permit expressionthereof. In separate embodiments, the cell is a bacterial cell, anamphibian cell, a yeast cell, an insect cell or a mammalian cell. Inanother embodiment, the vector is a baculovirus. In one embodiment, thevector is a plasmid.

This invention provides a plasmid designated pEXJ.HR-TL231 (ATCCAccession No. 203197). This plasmid comprises the regulatory elementsnecessary for expression of DNA in a mammalian cell operatively linkedto DNA encoding the human MCH1 receptor so as to permit expressionthereof.

This plasmid (pEXJ.HR-TL231) was deposited on Sep. 17, 1998, with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. 20852, U.S.A. under the provisions of the Budapest Treatyfor the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure and was accorded ATCC Accession No.203197.

This invention further provides for any vector or plasmid whichcomprises modified untranslated sequences, which are beneficial forexpression in desired host cells or for use in binding or functionalassays. For example, a vector or plasmid with untranslated sequences ofvarying lengths may express differing amounts of the polypeptidedepending upon the host cell used. In an embodiment, the vector orplasmid comprises the coding sequence of the polypeptide and theregulatory elements necessary for expression in the host cell.

This invention provides a cell comprising a vector comprising a nucleicacid encoding the human MCH1 receptor. In an embodiment, the cell is anon-mammalian cell. In a further embodiment, the non-mammalian cell is aXenopus oocyte cell or a Xenopus melanophore cell. In anotherembodiment, the cell is a mammalian cell. In a further embodiment, themammalian cell is a COS-7 cell, a 293 human embryonic kidney cell, aNIH-3T3 cell, a LM(tk-) cell, a mouse Y1 cell, or a CHO cell.

This invention provides an insect cell comprising a vector adapted forexpression in an insect cell which comprises a nucleic acid encoding ahuman MCH1 receptor. In another embodiment, the insect cell is an Sf9cell, an Sf21 cell or a Trichoplusia ni 5B1-4 (HighFive) cell.

This invention provides a membrane preparation isolated from any one ofthe cells described above.

This invention provides a nucleic acid probe comprising at least 15nucleotides, which probe specifically hybridizes with a nucleic acidencoding a human MCH1 receptor, wherein the probe has a unique sequencecorresponding to a sequence present within one of the two strands of thenucleic acid encoding a human MCH1 receptor present in plasmidpEXJ.HR-TL231. This invention also provides a nucleic acid probecomprising at least 15 nucleotides, which probe specifically hybridizeswith a nucleic acid encoding a human MCH1 receptor, wherein the probehas a unique sequence corresponding to a sequence present within (a) thenucleic acid sequence shown in FIG. 1 (Seq. I.D. No. 1) or (b) thereverse complement thereto. In one embodiment, the nucleic acid is DNA.In another embodiment, the nucleic acid is RNA.

As used herein, the phrase “specifically hybridizing” means the abilityof a nucleic acid molecule to recognize a nucleic acid sequencecomplementary to its own and to form double-helical segments throughhydrogen bonding between complementary base pairs.

Nucleic acid probe technology is well known to those skilled in the artwho will readily appreciate that such probes may vary greatly in lengthand may be labeled with a detectable label, such as a radioisotope orflourescent dye, to facilitate detection of the probe. DNA probemolecules may be produced by insertion of a DNA molecule which encodesthe polypeptides of this invention into suitable vectors, such asplasmids or bacteriophages, followed by transforming into suitablebacterial host cells, replication in the transformed bacterial hostcells and harvesting of the DNA probes, using methods well known in theart. Alternatively, probes may be generated chemically from DNAsynthesizers.

RNA probes may be generated by inserting the DNA molecule which encodesthe polypeptides of this invention downstream of a bacteriophagepromoter such as T3, T7, or SP6. Large amounts of RNA probe may beproduced by incubating the labeled nucleotides with the linearizedfragment where it contains an upstream promoter in the presence of theappropriate RNA polymerase.

This invention provides an antisense oligonucleotide having a sequencecapable of specifically hybridizing to RNA encoding a human MCH1receptor, so as to prevent translation of the RNA. This invention alsoprovides an antisense oligonucleotide having a sequence capable ofspecifically hybridizing to genomic DNA encoding a human MCH1 receptor.In one embodiment, the oligonucleotide comprises chemically modifiednucleotides or nucleotide analogues.

This invention provides an antibody capable of binding to a human MCH1receptor encoded by a nucleic acid encoding a human MCH1 receptor. Thisinvention also provides an agent capable of competitively inhibiting thebinding of the antibody to a human MCH1 receptor. In one embodiment, theantibody is a monoclonal antibody or antisera.

This invention provides a pharmaceutical composition comprising (a) anamount of the oligonucleotide capable of passing through a cell membraneand effective to reduce expression of a human MCH1 receptor and (b) apharmaceutically acceptable carrier capable of passing through the cellmembrane. In an embodiment, the oligonucleotide is coupled to asubstance which inactivates mRNA. In a further embodiment, the substancewhich inactivates mRNA is a ribozyme. In another embodiment, thepharmaceutically acceptable carrier comprises a structure which binds toa human MCH1 receptor on a cell capable of being taken up by the cellsafter binding to the structure. In a further embodiment, thepharmaceutically acceptable carrier is capable of binding to a humanMCH1 receptor which is specific for a selected cell type.

This invention provides a pharmaceutical composition which comprises anamount of an antibody effective to block binding of a ligand to a humanMCH1 receptor and a pharmaceutically acceptable carrier.

As used herein, the phrase “pharmaceutically acceptable carrier” meansany of the standard pharmaceutically acceptable carriers. Examplesinclude, but are not limited to, phosphate buffered saline,physiological saline, water, and emulsions, such as oil/water emulsions.

This invention provides a transgenic, nonhuman mammal expressing DNAencoding a human MCH1 receptor. This invention also provides atransgenic, nonhuman mammal comprising a homologous recombinationknockout of the native human MCH1 receptor. This invention furtherprovides a transgenic, nonhuman mammal whose genome comprises antisenseDNA complementary to the DNA encoding a human MCH1 receptor so placedwithin the genome as to be transcribed into antisense mRNA which iscomplementary to mRNA encoding the human MCH1 receptor and whichhybridizes to mRNA encoding the human MCH1 receptor, thereby reducingits translation. In an embodiment, the DNA encoding the human MCH1receptor additionally comprises an inducible promoter. In anotherembodiment, the DNA encoding the human MCH1 receptor additionallycomprises tissue specific regulatory elements. In a further embodiment,the transgenic, nonhuman mammal is a mouse.

Animal model systems which elucidate the physiological and behavioralroles of the polypeptides of this invention are produced by creatingtransgenic animals in which the activity of the polypeptide is eitherincreased or decreased, or the amino acid sequence of the expressedpolypeptide is altered, by a variety of techniques. Examples of thesetechniques include, but are not limited to: 1) Insertion of normal ormutant versions of DNA encoding the polypeptide, by microinjection,electroporation, retroviral transfection or other means well known tothose in the art, into appropriate fertilized embryos in order toproduce a transgenic animal or 2) Homologous recombination of mutant ornormal, human or animal versions of these genes with the native genelocus in transgenic animals to alter the regulation of expression or thestructure of these polypeptide sequences. The technique of homologousrecombination is well known in the art. It replaces the native gene withthe inserted gene and so is useful for producing an animal that cannotexpress native polypeptides but does express, for example, an insertedmutant polypeptide, which has replaced the native polypeptide in theanimal's genome by recombination, resulting in underexpression of thetransporter. Microinjection adds genes to the genome, but does notremove them, and so is useful for producing an animal which expressesits own and added polypeptides, resulting in overexpression of thepolypeptides.

One means available for producing a transgenic animal, with a mouse asan example, is as follows: Female mice are mated, and the resultingfertilized eggs are dissected out of their oviducts. The eggs are storedin an appropriate medium such as M2 medium. DNA or cDNA encoding apolypeptide of this invention is purified from a vector by methods wellknown in the art. Inducible promoters may be fused with the codingregion of the DNA to provide an experimental means to regulateexpression of the trans-gene. Alternatively, or in addition, tissuespecific regulatory elements may be fused with the coding region topermit tissue-specific expression of the trans-gene. The DNA, in anappropriately buffered solution, is put into a microinjection needle(which may be made from capillary tubing using a pipette puller) and theegg to be injected is put in a depression slide. The needle is insertedinto the pronucleus of the egg, and the DNA solution is injected. Theinjected egg is then transferred into the oviduct of a pseudopregnantmouse (a mouse stimulated by the appropriate hormones to maintainpregnancy but which is not actually pregnant), where it proceeds to theuterus, implants, and develops to term. As noted above, microinjectionis not the only method for inserting DNA into the egg cell, and is usedhere only for exemplary purposes.

This invention provides a process for identifying a chemical compoundwhich specifically binds to a mammalian MCH1 receptor which comprisescontacting cells comprising DNA encoding, and expressing on their cellsurface, the mammalian MCH1 receptor, with the compound under conditionssuitable for binding, and detecting specific binding of the chemicalcompound to the mammalian MCH1 receptor, wherein the cells do notnormally express the mammalian MCH1 receptor and the DNA encoding themammalian MCH1 receptor (a) hybridizes to a nucleic acid having thedefined sequence shown in FIG. 1 (Seq. ID No. 1) under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a MCH1 ligand is added to the culture and the CHO cellscontain the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement. This invention also provides aprocess for identifying a chemical compound which specifically binds toa mammalian MCH1 receptor which comprises contacting a membranepreparation from cells comprising DNA encoding, and expressing on theircell surface, the mammalian MCH1 receptor, with the compound underconditions suitable for binding, and detecting specific binding of thechemical compound to the mammalian MCH1 receptor, wherein the cells donot normally express the mammalian MCH1 receptor and the DNA encodingthe mammalian MCH1 receptor (a) hybridizes to a nucleic acid having thedefined sequence shown in FIG. 1 (Seq. ID No. 1) under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a MCH1 ligand is added to the culture and the CHO cellscontain the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement. In one embodiment, the MCH1 receptoris a human MCH1 receptor. In another embodiment, the MCH1 receptor is arat MCH1 receptor. In another embodiment, the mammalian MCH1 receptorcomprises substantially the same amino acid sequence as the sequence ofthe human MCH1 receptor encoded by plasmid pEXJ.HR-TL231. In a furtherembodiment, the mammalian MCH1 receptor comprises substantially the sameamino acid sequence as that shown in FIG. 2 (Seq. ID No. 2). In anotherembodiment, the mammalian MCH1 receptor comprises the amino acidsequence shown in FIG. 2 (Seq. ID No. 2). In one embodiment, thecompound is not previously known to bind to a mammalian MCH1 receptor.This invention further provides a compound identified by theabove-described processes.

In one embodiment of the above-described processes, the cell is aninsect cell. In another embodiment, the cell is a mammalian cell. In afurther embodiment, the cell is nonneuronal in origin. In a furtherembodiment, the nonneuronal cell is a COS-7 cell, 293 human embryonickidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1 cell, or a LM(tk-)cell.

This invention provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a mammalianMCH1 receptor which comprises contacting cells expressing on their cellsurface the mammalian MCH1 receptor, with both the chemical compound anda second chemical compound known to bind to the receptor, and separatelywith only the second chemical compound, under conditions suitable forbinding of both compounds, and detecting specific binding of thechemical compound to the mammalian MCH1 receptor, a decrease in thebinding of the second chemical compound to the mammalian MCH1 receptorin the presence of the chemical compound indicating that the chemicalcompound binds to the mammalian MCH1 receptor, wherein the cells do notnormally express the mammalian MCH1 receptor and the DNA encoding themammalian MCH1 receptor (a) hybridizes to a nucleic acid having thedefined sequence shown in FIG. 1 (Seq. ID No. 1) under low stringencyconditions or a sequence complementary thereto and (b) is furthercharacterized by its ability to cause a change in the pH of a culture ofCHO cells when a MCH1 ligand is added to the culture and the CHO cellscontain the nucleic acid which hybridized to the nucleic acid having thedefined sequence or its complement.

This invention also provides a process involving competitive binding foridentifying a chemical compound which specifically binds to a mammalianMCH1 receptor which comprises contacting a membrane preparation fromcells expressing on their cell surface the mammalian MCH1 receptor, withboth the chemical compound and a second chemical compound known to bindto the receptor, and separately with only the second chemical compound,under conditions suitable for binding of both compounds, and detectingspecific binding of the chemical compound to the mammalian MCH1receptor, a decrease in the binding of the second chemical compound tothe mammalian MCH1 receptor in the presence of the chemical compoundindicating that the chemical compound binds to the mammalian MCH1receptor, wherein the cells do not normally express the mammalian MCH1receptor and the DNA encoding the mammalian MCH1 receptor (a) hybridizesto a nucleic acid having the defined sequence shown in FIG. 1 (Seq. IDNo. 1) under low stringency conditions or a sequence complementarythereto and (b) is further characterized by its ability to cause achange in the pH of a culture of CHO cells when a MCH1 ligand is addedto the culture and the CHO cells contain the nucleic acid whichhybridized to the nucleic acid having the defined sequence or itscomplement.

In one embodiment, the mammalian MCH1 receptor is a human MCH1 receptor.In another embodiment, the mammalian MCH1 receptor is a rat MCH1receptor. In another embodiment, the mammalian MCH1 receptor comprisessubstantially the same amino acid sequence as the human MCH1 receptorencoded by plasmid PEXJ.HR-TL231. In a further embodiment, the mammalianMCH1 receptor comprises substantially the same amino acid sequence asthat shown in FIG. 2 (Seq. I.D. No. 2). In another embodiment, themammalian MCH1 receptor comprises the amino acid sequence shown in FIG.2 (Seq. I.D. No. 2).

In one embodiment, the cell is an insect cell. In another embodiment,the cell is a mammalian cell. In a further embodiment, the cell isnonneuronal in origin. In another embodiment, the nonneuronal cell is aCOS-7 cell, 293 human embryonic kidney cell, a CHO cell, a NIH-3T3 cell,a mouse Y1 cell, or a LM(tk-) cell. In one embodiment, the compound isnot previously known to bind to a mammalian MCH1 receptor.

This invention provides a compound identified by the above-describedprocesses.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian MCH1 receptor to identify acompound which specifically binds to the mammalian MCH1 receptor, whichcomprises (a) contacting cells transfected with and expressing DNAencoding the mammalian MCH1 receptor with the plurality of compounds notknown to bind specifically to the mammalian MCH1 receptor, underconditions permitting binding of compounds known to bind the mammalianMCH1 receptor; (b) determining whether the binding of a compound knownto bind to the mammalian MCH1 receptor is reduced in the presence of thecompounds within the plurality of compounds, relative to the binding ofthe compound in the absence of the plurality of compounds; and if so (c)separately determining the binding to the mammalian MCH1 receptor ofcompounds included in the plurality of compounds, so as to therebyidentify the compound which specifically binds to the mammalian MCH1receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to bind to a mammalian MCH1 receptor to identify acompound which specifically binds to the mammalian MCH1 receptor, whichcomprises (a) contacting a membrane preparation from cells transfectedwith and expressing the mammalian MCH1 receptor with the plurality ofcompounds not known to bind specifically to the mammalian MCH1 receptor,under conditions permitting binding of compounds known to bind themammalian MCH1 receptor; (b) determining whether the binding of acompound known to bind to the mammalian MCH1 receptor is reduced in thepresence of the compounds within the plurality of compounds, relative tothe binding of the compound in the absence of the plurality ofcompounds; and if so (c) separately determining the binding to themammalian MCH1 receptor of compounds included in the plurality ofcompounds, so as to thereby identify the compound which specificallybinds to the mammalian MCH1 receptor.

In one embodiment of the above-described methods, the mammalian MCH1receptor is a human MCH1 receptor. In another embodiment, the mammalianMCH1 receptor is a rat MCHI receptor. In another embodiment, the cell isa mammalian cell. In a further embodiment, the mammalian cell isnon-neuronal in origin. In another embodiment, the non-neuronal cell isa COS-7 cell, a 293 human embryonic kidney cell, a LM(tk-) cell, a CHOcell, a mouse Y1 cell, or an NIH-3T3 cell.

This invention also provides a method of detecting expression of amammalian MCH1 receptor by detecting the presence of mRNA coding for themammalian MCH1 receptor which comprises obtaining total mRNA from thecell and contacting the mRNA so obtained from a nucleic acid probe underhybridizing conditions, detecting the presence of mRNA hybridizing tothe probe, and thereby detecting the expression of the mammalian MCH1receptor by the cell.

This invention further provides a method of detecting the presence of amammalian MCH1 receptor on the surface of a cell which comprisescontacting the cell with an antibody under conditions permitting bindingof the antibody to the receptor, detecting the presence of the antibodybound to the cell, and thereby detecting the presence of the mammalianMCH1 receptor on the surface of the cell.

This invention provides a method of determining the physiologicaleffects of varying levels of activity of human MCH1 receptors whichcomprises producing a transgenic, nonhuman mammal whose levels of humanMCH1 receptor activity are varied by use of an inducible promoter whichregulates human MCH1 receptor expression.

This invention also provides a method of determining the physiologicaleffects of varying levels of activity of human MCH1 receptors whichcomprises producing a panel of transgenic, nonhuman mammals eachexpressing a different amount of human MCH1 receptor.

This invention provides a method for identifying an antagonist capableof alleviating an abnormality wherein the abnormality is alleviated bydecreasing the activity of a human MCH1 receptor comprisingadministering a compound to a transgenic, nonhuman mammal, anddetermining whether the compound alleviates the physical and behavioralabnormalities displayed by the transgenic, nonhuman mammal as a resultof overactivity of a human MCH1 receptor, the alleviation of theabnormality identifying the compound as an antagonist. This inventionalso provides an antagonist identified by the above-described method.This invention further provides a pharmaceutical composition comprisingan antagonist identified by the above-described method and apharmaceutically acceptable carrier. This invention provides a method oftreating an abnormality in a subject wherein the abnormality isalleviated by decreasing the activity of a human MCH1 receptor whichcomprises administering to the subject an effective amount of thispharmaceutical composition, thereby treating the abnormality.

This invention provides a method for identifying an agonist capable ofalleviating an abnormality in a subject wherein the abnormality isalleviated by increasing the activity of a human MCH1 receptorcomprising administering a compound to transgenic, nonhuman mammal, anddetermining whether the compound alleviates the physical and behavioralabnormalities displayed by the transgenic, nonhuman mammal, thealleviation of the abnormality identifying the compound as an agonist.This invention also provides an agonist identified by theabove-described method. This invention further provides a pharmaceuticalcomposition comprising an agonist identified by the above-describedmethod and a pharmaceutically acceptable carrier. This invention furtherprovides a method of treating an abnormality in a subject wherein theabnormality is alleviated by increasing the activity of a human MCH1receptor which comprises administering to the subject an effectiveamount of this pharmaceutical composition, thereby treating theabnormality.

This invention provides a method for diagnosing a predisposition to adisorder associated with the activity of a specific mammalian allelewhich comprises: (a) obtaining DNA of subjects suffering from thedisorder; (b) performing a restriction digest of the DNA with a panel ofrestriction enzymes; (c) electrophoretically separating the resultingDNA fragments on a sizing gel; (d) contacting the resulting gel with anucleic acid probe capable of specifically hybridizing with a uniquesequence included within the sequence of a nucleic acid moleculeencoding a human MCH1 receptor and labeled with a detectable marker; (e)detecting labeled bands which have hybridized to the DNA encoding ahuman MCH1 receptor labeled with a detectable marker to create a uniqueband pattern specific to the DNA of subjects suffering from thedisorder; (f) preparing DNA obtained for diagnosis by steps (a)-(e); and(g) comparing the unique band pattern specific to the DNA of subjectssuffering from the disorder from step (e) and the DNA obtained fordiagnosis from step (f) to determine whether the patterns are the sameor different and to diagnose thereby predisposition to the disorder ifthe patterns are the same. In one embodiment, a disorder associated withthe activity of a specific mammalian allele is diagnosed.

This invention provides a method of preparing the purified human MCH1receptor which comprises: (a) inducing cells to express the human MCH1receptor; (b) recovering the human MCH1 receptor from the induced cells;and (c) purifying the human MCH1 receptor so recovered.

This invention provides a method of preparing the purified human MCH1receptor which comprises: (a) inserting nucleic acid encoding the humanMCH1 receptor in a suitable vector; (b) introducing the resulting vectorin a suitable host cell; (c) placing the resulting cell in suitablecondition permitting the production of the isolated human MCH1 receptor;(d) recovering the human MCH1 receptor produced by the resulting cell;and (e) purifying the human MCH1 receptor so recovered.

This invention provides a process for determining whether a chemicalcompound is a mammalian MCH1 receptor agonist which comprises contactingcells transfected with and expressing DNA encoding the mammalian MCH1receptor with the compound under conditions permitting the activation ofthe mammalian MCH1 receptor, and detecting an increase in mammalian MCH1receptor activity, so as to thereby determine whether the compound is amammalian MCH1 receptor agonist. This invention also provides a processfor determining whether a chemical compound is a mammalian MCH1 receptorantagonist which comprises contacting cells transfected with andexpressing DNA encoding the mammalian MCH1 receptor with the compound inthe presence of a known mammalian MCH1 receptor agonist, underconditions permitting the activation of the mammalian MCH1 receptor, anddetecting a decrease in mammalian MCH1 receptor activity, so as tothereby determine whether the compound is a mammalian MCH1 receptorantagonist. In one embodiment, the mammalian MCH1 receptor is a humanMCH1 receptor.

This invention further provides a pharmaceutical composition whichcomprises an amount of a mammalian MCH1 receptor agonist determined bythe above-described process effective to increase activity of amammalian MCH1 receptor and a pharmaceutically acceptable carrier. Inone embodiment, the mammalian MCH1 receptor agonist is not previouslyknown.

This invention provides a pharmaceutical composition which comprises anamount of a mammalian MCH1 receptor antagonist determined by theabove-described process effective to reduce activity of a mammalian MCH1receptor and a pharmaceutically acceptable carrier. In one embodiment,the mammalian MCH1 receptor antagonist is not previously known.

This invention provides a process for determining whether a chemicalcompound specifically binds to and activates a mammalian MCH1 receptor,which comprises contacting cells producing a second messenger responseand expressing on their cell surface the mammalian MCH1 receptor,wherein such cells do not normally express the mammalian MCH1 receptor,with the chemical compound under conditions suitable for activation ofthe mammalian MCH1 receptor, and measuring the second messenger responsein the presence and in the absence of the chemical compound, a change inthe second messenger response in the presence of the chemical compoundindicating that the compound activates the mammalian MCH1 receptor. Inone embodiment, the second messenger response comprises chloride channelactivation and the change in second messenger is an increase in thelevel of inward chloride current.

This invention also provides a process for determining whether achemical compound specifically binds to and inhibits activation of amammalian MCH1 receptor, which comprises separately contacting cellsproducing a second messenger response and expressing on their cellsurface the mammalian MCH1 receptor, wherein such cells do not normallyexpress the mammalian MCH1 receptor, with both the chemical compound anda second chemical compound known to activate the mammalian MCH1receptor, and with only the second chemical compound, under conditionssuitable for activation of the mammalian MCH1 receptor, and measuringthe second messenger response in the presence of only the secondchemical compound and in the presence of both the second chemicalcompound and the chemical compound, a smaller change in the secondmessenger response in the presence of both the chemical compound and thesecond chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the mammalian MCH1 receptor. In one embodiment, the secondmessenger response comprises chloride channel activation and the changein second messenger response is a smaller increase in the level ofinward chloride current in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound. This invention also provides the above-describedprocesses performed with membrane preparations from cells producing asecond messenger response and transfected with and expressing themammalian MCH1 receptor.

In one embodiment of the above-described processes, the mammalian MCH1receptor is a human MCH1 receptor. In another embodiment, the mammalianMCH1 receptor is a rat MCH1 receptor. In another embodiment, themammalian MCH1 receptor comprises substantially the same amino acidsequence as encoded by the plasmid pEXJ.HR-TL231. In a furtherembodiment, the mammalian MCH1 receptor comprises substantially the sameamino acid sequence as that shown in FIG. 2 (Seq. I.D. No. 2). Inanother embodiment, the mammalian MCH1 receptor comprises an amino acidsequence as shown in FIG. 2 (Seq. I.D. No. 2). In an embodiment, thecell is an insect cell. In a further embodiment, the cell is a mammaliancell. In a still further embodiment, the mammalian cell is nonneuronalin origin. In another embodiment, the nonneuronal cell is a COS-7 cell,CHO cell, 293 human embryonic kidney cell, NIH-3T3 cell or LM(tk-) cell.In an embodiment, the compound is not previously known to bind to amammalian MCH1 receptor. This invention also provides a compounddetermined by the above-described processes.

This invention also provides a pharmaceutical composition whichcomprises an amount of a mammalian MCH1 receptor agonist determined bythe above-described processes effective to increase activity of amammalian MCH1 receptor and a pharmaceutically acceptable carrier. Inone embodiment, the mammalian MCH1 receptor agonist is not previouslyknown.

This invention further provides a pharmaceutical composition whichcomprises an amount of a mammalian MCH1 receptor antagonist determinedby the above-described processes effective to reduce activity of amammalian MCH1 receptor and a pharmaceutically acceptable carrier. Inone embodiment, the mammalian MCH1 receptor antagonist is not previouslyknown.

This invention provides a method of screening a plurality of chemicalcompounds not known to activate a mammalian MCH1 receptor to identify acompound which activates the mammalian MCH1 receptor which comprises:(a) contacting cells transfected with and expressing the mammalian MCH1receptor with the plurality of compounds not known to activate themammalian MCH1 receptor, under conditions permitting activation of themammalian MCH1 receptor; (b) determining whether the activity of themammalian MCH1 receptor is increased in the presence of the compounds;and if so (c) separately determining whether the activation of themammalian MCH1 receptor is increased by each compound included in theplurality of compounds, so as to thereby identify the compound whichactivates the mammalian MCH1 receptor. In one embodiment, the mammalianMCH1 receptor is a human MCH1 receptor. In another embodiment, themammalian MCH1 receptor is a rat MCH1 receptor.

This invention provides a method of screening a plurality of chemicalcompounds not known to inhibit the activation of a mammalian MCH1receptor to identify a compound which inhibits the activation of themammalian MCH1 receptor, which comprises: (a) contacting cellstransfected with and expressing the mammalian MCH1 receptor with theplurality of compounds in the presence of a known mammalian MCH1receptor agonist, under conditions permitting activation of themammalian MCH1 receptor; (b) determining whether the activation of themammalian MCH1 receptor is reduced in the presence of the plurality ofcompounds, relative to the activation of the mammalian MCH1 receptor inthe absence of the plurality of compounds; and if so (c) separatelydetermining the inhibition of activation of the mammalian MCH1 receptorfor each compound included in the plurality of compounds, so as tothereby identify the compound which inhibits the activation of themammalian MCH1 receptor. In one embodiment, the mammalian MCH1 receptoris a human MCH1 receptor. In another embodiment, the mammalian MCH1receptor is a rat MCH1 receptor.

In one embodiment of the above-described methods, the cell is amammalian cell. In another embodiment, the mammalian cell isnon-neuronal in origin. In a further embodiment, the non-neuronal cellis a COS-7 cell, a 293 human embryonic kidney cell, a LM(tk-) cell or anNIH-3T3 cell.

This invention provides a pharmaceutical composition comprising acompound identified by the above-described methods effective to increasemammalian MCH1 receptor activity and a pharmaceutically acceptablecarrier.

This invention also provides a pharmaceutical composition comprising acompound identified by the above-described methods effective to decreasemammalian MCH1 receptor activity and a pharmaceutically acceptablecarrier.

This invention further provides a method of measuring receptoractivation in an oocyte expression system such as a Xenopus oocyteexpression system or melanophore. In an embodiment, receptor activationis determined by measurement of ion channel activity. In anotherembodiment, receptor activation is measured by aequorin luminescence.

Expression of genes in Xenopus oocytes is well known in the art(Coleman, A., 1984; Masu, Y.,et al., 1994) and is performed usingmicroinjection of native mRNA or in vitro synthesized mRNA into frogoocytes. The preparation of in vitro synthesized mRNA can be performedby various standard techniques (Sambrook, et al. 1989) including usingT7 polymerase with the mCAP RNA mapping kit (Stratagene).

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by increasing the activity of amammalian MCH1 receptor which comprises administering to the subject anamount of a compound which is a mammalian MCH1 receptor agonisteffective to treat the abnormality. In separate embodiments, theabnormality is a regulation of a steroid or pituitary hormone disorder,an epinephrine release disorder, a gastrointestinal disorder, acardiovascular disorder, an electrolyte balance disorder, hypertension,diabetes, a respiratory disorder, asthma, a reproductive functiondisorder, an immune disorder, an endocrine disorder, a musculoskeletaldisorder, a neuroendocrine disorder, a cognitive disorder, a memorydisorder, a sensory modulation and transmission disorder, a motorcoordination disorder, a sensory integration disorder, a motorintegration disorder, a dopaminergic function disorder, a sensorytransmission disorder, an olfaction disorder, a sympathetic innervationdisorder, an affective disorder, a stress-related disorder, afluid-balance disorder, a seizure disorder, pain, psychotic behavior,morphine tolerance, opiate addiction or migraine.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is alleviated by decreasing the activity of amammalian MCH1 receptor which comprises administering to the subject anamount of a compound which is a mammalian MCH1 receptor antagonisteffective to treat the abnormality. In separate embodiments, theabnormality is a regulation of a steroid or pituitary hormone disorder,an epinephrine release disorder, a gastrointestinal disorder, acardiovascular disorder, an electrolyte balance disorder, hypertension,diabetes, a respiratory disorder, asthma, a reproductive functiondisorder, an immune disorder, an endocrine disorder, a musculoskeletaldisorder, a neuroendocrine disorder, a cognitive disorder, a memorydisorder, a sensory modulation and transmission disorder, a motorcoordination disorder, a sensory integration disorder, a motorintegration disorder, a dopaminergic function disorder, a sensorytransmission disorder, an olfaction disorder, a sympathetic innervationdisorder, an affective disorder, a stress-related disorder, afluid-balance disorder, a seizure disorder, pain, psychotic behavior,morphine tolerance, opiate addiction or migraine.

This invention provides a process for making a composition of matterwhich specifically binds to a mammalian MCH1 receptor which comprisesidentifying a chemical compound using any of the processes describedherein for identifying a compound which binds to and/or activates orinhibits activation of a mammalian MCH1 receptor and then synthesizingthe chemical compound or a novel structural and functional analog orhomolog thereof. In one embodiment, the mammalian MCH1 receptor is ahuman MCH1 receptor. In another embodiment, the mammalian MCH1 receptoris a rat MCH1 receptor.

This invention further provides a process for preparing a pharmaceuticalcomposition which comprises admixing a pharmaceutically acceptablecarrier and a therapeutically effective amount of a chemical compoundidentified by any of the processes described herein for identifying acompound which binds to and/or activates or inhibits activation of amammalian MCH1 receptor or a novel structural and functional analog orhomolog thereof. In one embodiment, the mammalian MCH1 receptor is ahuman MCH1 receptor. In another embodiment, the mammalian MCH1 receptoris a rat MCH1 receptor.

Thus, once the gene for a targeted receptor subtype is cloned, it isplaced into a recipient cell which then expresses the targeted receptorsubtype on its surface. This cell, which expresses a single populationof the targeted human receptor subtype, is then propagated resulting inthe establishment of a cell line. This cell line, which constitutes adrug discovery system, is used in two different types of assays: bindingassays and functional assays. In binding assays, the affinity of acompound for both the receptor subtype that is the target of aparticular drug discovery program and other receptor subtypes that couldbe associated with side effects are measured. These measurements enableone to predict the potency of a compound, as well as the degree ofselectivity that the compound has for the targeted receptor subtype overother receptor subtypes. The data obtained from binding assays alsoenable chemists to design compounds toward or away from one or more ofthe relevant subtypes, as appropriate, for optimal therapeutic efficacy.In functional assays, the nature of the response of the receptor subtypeto the compound is determined. Data from the functional assays showwhether the compound is acting to inhibit or enhance the activity of thereceptor subtype, thus enabling pharmacologists to evaluate compoundsrapidly at their ultimate human receptor subtypes targets permittingchemists to rationally design drugs that will be more effective and havefewer or substantially less severe side effects than existing drugs.

Approaches to designing and synthesizing receptor subtype-selectivecompounds are well known and include traditional medicinal chemistry andthe newer technology of combinatorial chemistry, both of which aresupported by computer-assisted molecular modeling. With such approaches,chemists and pharmacologists use their knowledge of the structures ofthe targeted receptor subtype and compounds determined to bind and/oractivate or inhibit activation of the receptor subtype to design andsynthesize structures that will have activity at these receptorsubtypes.

Combinatorial chemistry involves automated synthesis of a variety ofnovel compounds by assembling them using different combinations ofchemical building blocks. The use of combinatorial chemistry greatlyaccelerates the process of generating compounds. The resulting arrays ofcompounds are called libraries and are used to screen for compounds(“lead compounds”) that demonstrate a sufficient level of activity atreceptors of interest. Using combinatorial chemistry it is possible tosynthesize “focused” libraries of compounds anticipated to be highlybiased toward the receptor target of interest.

Once lead compounds are identified, whether through the use ofcombinatorial chemistry or traditional medicinal chemistry or otherwise,a variety of homologs and analogs are prepared to facilitate anunderstanding of the relationship between chemical structure andbiological or functional activity. These studies define structureactivity relationships which are then used to design drugs with improvedpotency, selectivity and pharmacokinetic properties. Combinatorialchemistry is also used to rapidly generate a variety of structures forlead optimization. Traditional medicinal chemistry, which involves thesynthesis of compounds one at a time, is also used for furtherrefinement and to generate compounds not accessible by automatedtechniques. Once such drugs are defined the production is scaled upusing standard chemical manufacturing methodologies utilized throughoutthe pharmaceutical and chemistry industry.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS Materials and Methods

Cloning of Human MCH1 Receptor

Discovery of an Expressed Sequence Tag (EST) F07228 in GENEMBLHomologous to FB41a

A BLAST search of GENEMBL was performed with the GCG sequence analysispackage (Genetics Computer Group, Madison, Wis.) using a SynapticPharmaceutical Corporation proprietary sequence, FB41a, as a query. Thisresulted in the identification of an EST (accession number F07228) witha high degree of homology to FB41a and somatostatin, opiate and galaninreceptors.

Construction and Screening of a Human Hippocampal cDNA Library

Poly A+ RNA was purified from human hippocampal RNA (Clontech) using aFastTrack kit (Invitrogen, Corp.). DS− cDNA was synthesized from poly A+RNA according to Gubler and Hoffman (1983) with minor modifications. Theresulting cDNA was ligated to BstXI adaptors (Invitrogen, Corp.) and theexcess adaptors removed by exclusion column chromatography. Highmolecular weight fractions of size-selected ds-cDNA were ligated inpEXJ.BS, an Okayama and Berg expression vector modified from pcEXV(Miller and Germain, 1986) to contain BstXI and other additionalrestriction sites. A total of 2.2×10⁶independent clones with a meaninsert size of 3.0 kb were generated. The library was plated on agarplates (ampicillin selection) and glycerol stocks for 450 pools of 5000independent clones were prepared. Primary glycerol stocks were alsogrouped together in groups of approximately 10 to create superpools.

Cloning of the Full-Length Sequence of MCH1

Glycerol stocks of the superpools and primary pools from the humanhippocampal cDNA library were screened by PCR with F07228 specificprimers T579 and T580 using Taq DNA Polymerase (Boehringer-Mannheim,Indianapolis, Ind.) and the following PCR protocol: 94° C. hold for 5minutes; 40 cycles of 94° C. for 2 minute, 68° C. for 4 minutes; 7minute hold at 68° C.; 40° C. hold until the samples are run on a gel.One positive primary pool 490, was successively divided into subpools,amplified in LB medium overnight and screened by PCR using primers T579and T580. One positive subpool, 490-4-10-23 was plated on agar plates(ampicillin selection), and colonies were transferred to nitrocellulosemembranes (Schleicher and Schuell, Keene, N.H.). Filters were hybridizedfor two days under high stringency conditions with 10⁶ cpm/ml of a³²P-labeled cDNA probe, T581, designed against the F07228 EST sequence.Filters were washed and apposed to Biomax MS film (Kodak) Seven positivecolonies were picked, streaked on LB-AMP plates, and grown overnight.Two individual colonies from each of the original seven were picked andsubjected to vector-anchored PCR using the following primer pairs: T95,T580 and T94, T579. One positive colony, G1, was amplified overnight inTB and processed for plasmid purification. This plasmid was designatedTL230 and sequenced on both strands with a Sequenase kit (U.S.Biochemical, Cleveland, Ohio). Nucleotide and peptide sequence analysiswere performed with GCG programs (Genetics Computer Group, Madison,Wis.). A HindIII- KpnI fragment of TL230 was subcloned into themammalian expression vector PEXJ, and named TL231.

Primers and Probes

TL579: 5′-GGGAACTCCACGGTCATCTTCGCGGT-3′ (Seq. I.D. No. 5)

TL580: 5′-TAGCGGTCAATGGCCATGGCGGTCAG-3′ (Seq. I.D. No. 6)

TL581: 5′-CTCCTGGGCATGCCCTTCATGATCCACCAGCTCATGGGCAATGGG-3′ (Seq. I.D.No. 7)

TL94: 5′-CTTCTAGGCCTGTACGGAAGTGTTA-3′ (Seq. I.D. No. 8)

TL95: 5′-GTTGTGGTTTGTCCAAACTCATCAATG-3′ (Seq. I.D. No. 9)

Isolation of a Fragment of a Species Homologue of TL231 (Human MCH1)

To obtain a fragment of a species homologue of TL231, the speciesgenomic DNA (Clontech) may be amplified with a forward PCR primercorresponding to one of the TM regions of TL231 and a reverse primercorresponding to another TM region of TL231. PCR may be performed withthe Expand Long Template PCR System (Boeringer Mannheim), for example,under the following conditions: 30 sec at 94° C., 1.5 min at 50° C., 1.5min at 68° C. for 40 cycles, with a pre- and post-incubation of 5 min at94° C. and 7 min at 68° C., respectively. A band is isolated, subclonedusing the TA cloning kit (Invitrogen), and sequenced. The sequence isrun and analyzed on an ABI PRISM 377 BigDye Terminator Cycle SequencingKit Sequencer. Forward and reverse PCR primers are designed against thissequence and used to amplify a band from genomic DNA using, for example,the following conditions: 30 sec at 94° C., 1.5 min at 68° C. for 35cycles, with a pre- and post-incubation of 5 min at 94° C. and 5 min at68° C., respectively. The PCR product is subcloned using the TA cloningkit (Invitrogen). Miniprep cultures of transformants are prepared andsequenced as above.

Isolation of a Full-Length Species Homolog of TL231 (Human MCH1)

A nucleic acid sequence encoding an MCH1 receptor may be isolated usingstandard molecular biology techniques and approaches such as thosebriefly described below:

Approach #1: To obtain a full-length MCH1 receptor, a cosmid librarycould be screened with a ³²P-labeled oligonucleotide probe.

The full-length sequence may be obtained by sequencing this cosmid clonewith additional sequencing primers. Since one intron is present in thisgene the full-length intronless gene may be obtained from cDNA usingstandard molecular biology techniques. For example, a forward PCR primerdesigned in the 5′UT and a reverse PCR primer designed in the 3′UT maybe used to amplify a full-length, intronless gene from cDNA. Standardmolecular biology techniques could be used to subclone this gene into amammalian expression vector.

Approach #2: Standard molecular biology techniques could be used toscreen commercial cDNA phage libraries by hybridization under highstringency with a ³²P-labeled oligonucleotide probe. One may isolate afull-length MCH1 receptor by obtaining a plaque purified clone from thelambda libraries and then subjecting the clone to direct DNA sequencing.Alternatively, standard molecular biology techniques could be used toscreen in-house cDNA plasmid libraries by PCR amplification of librarypools using primers to the MCH1 sequence. A full-length clone could beisolated by Southern hybridization of colony lifts of positive poolswith a ³²P-labeled oligonucleotide probe.

Approach #3: As yet another alternative method, one could utilize 3′ and5′ RACE to generate PCR products from cDNA expressing MCH1 which containthe additional sequences of MCH1. These RACE PCR products could then besequenced to determine the missing sequence. This new sequence couldthen be used to design a forward PCR primer in the 5′UT and a reverseprimer in the 3′UT. These primers could then be used to amplify afull-length MCH1 clone from cDNA.

Host Cells

A broad variety of host cells can be used to study heterologouslyexpressed proteins. These cells include but are not restricted toassorted mammalian lines such as; Cos-7, CHO, LM(tk-), HEK293, etc.;insect cell lines such as; Sf9, Sf21, etc.; amphibian cells such asxenopus oocytes; and others.

COS-7 cells are grown on 150 mm plates in DMEM with supplements(Dulbecco's Modified Eagle Medium with 10% bovine calf serum, 4 mMglutamine, 100 units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5%CO₂. Stock plates of COS-7 cells are trypsinized and split 1:6 every 3-4days.

Human embryonic kidney 293 cells are grown on 150 mm plates in DMEM withsupplements (10% bovine calf serum, 4 mM glutamine, 100 units/mlpenicillin/100 μg/ml streptomycin) at 37° C., 5% CO₂. Stock plates of293 cells are trypsinized and split 1:6 every 3-4 days.

Mouse fibroblast LM(tk-) cells are grown on 150 mm plates in D-MEM withsupplements (Dulbecco's Modified Eagle Medium with 10% bovine calfserum, 4 mM glutamine, 100 units/ml penicillin/100 μg/ml streptomycin)at 37° C., 5% CO_(2.) Stock plates of LM(tk-) cells are trypsinized andsplit 1:10 every 3-4 days.

Chinese hamster ovary (CHO) cells were grown on 150 mm plates in HAM'sF-12 medium with supplements (10% bovine calf serum, 4 mM L-glutamineand 100 units/ml penicillin/100 μg/ml streptomycin) at 37° C., 5% CO₂.Stock plates of CHO cells are trypsinized and split 1:8 every 3-4 days.

Mouse embryonic fibroblast NIH-3T3 cells are grown on 150 mm plates inDulbecco's Modified Eagle Medium (DMEM) with supplements (10% bovinecalf serum, 4 mM glutamine, 100 units/ml penicillin/100 μg/mlstreptomycin) at 37° C., 5% CO₂. Stock plates of NIH-3T3 cells aretrypsinized and split 1:15 every 3-4 days.

Sf9 and Sf21 cells are grown in monolayers on 150 mm tissue culturedishes in TMN-FH media supplemented with 10% fetal calf serum, at 27°C., no CO₂. High Five insect cells are grown on 150 mm tissue culturedishes in Ex-Cell 400™ medium supplemented with L-Glutamine, also at 27°C., no CO₂.

In some cases, cell lines that grow as adherent monolayers can beconverted to suspension culture to increase cell yield and provide largebatches of uniform assay material for routine receptor screeningprojects.

Xenopus oocytes can also be used as a host system for transientexpression of heterologous proteins. Their maintenance and usage isdescribed in the electrophysiological methods section that follows.

Transient Expression

DNA encoding proteins to be studied can be transiently expressed in avariety of mammalian, insect, amphibian and other cell lines by severalmethods including but not restricted to; calcium phosphate-mediated,DEAE-dextran mediated, Liposomal-mediated, viral-mediated,electroporation-mediated and microinjection delivery. Each of thesemethods may require optimization of assorted experimental parametersdepending on the DNA, cell line, and the type of assay to besubsequently employed.

A typical protocol for the calcium phosphate method as applied toLM(tk-) cells is described as follows; Adherent cells are harvestedapproximately twenty-four hours before transfection and replated at adensity of 1-2×10⁵ cells/cm² in a 100 mm tissue culture dish and allowedto incubate over night at 37° C. at 5% CO₂. 250 μl of a mixture of CaCl₂and DNA (20 μg DNA in 250 mM CaCl₂) is added to a 5 ml plastic tube and250 μl of 2×HBS (250 mM NaCl, 10 mM KCl, 1.5 mM Na₂HPO₄, 12 mM dextrose,50 mM HEPES) is slowly added with gentle mixing. The mixture is allowedto incubate for 20 minutes at room temperature to allow a DNAprecipitate to form. The cells are then washed with complete medium, 10ml of culture medium is added to each plate, followed by addition of theDNA precipitate. The cells are then incubated for 24 to 48 hours at 37°C. at 5% CO₂.

A typical protocol for the DEAE-dextran method as applied to Cos-7 cellsis described as follows; Cells to be used for transfection are split 24hours prior to the transfection to provide flasks which are 70-80%confluent at the time of transfection. Briefly, 8 μg of receptor DNAplus 8 μg of any additional DNA needed (e.g. G₆₀ protein expressionvector, reporter construct, antibiotic resistance marker, mock vector,etc.) are added to 9 ml of complete DMEM plus DEAE-dextran mixture (10mg/ml in PBS). Cos-7 cells plated into a T225 flask (sub-confluent) arewashed once with PBS and the DNA mixture is added to each flask. Thecells are allowed to incubate for 30 minutes at 37° C., 5% CO₂.Following the incubation, 36 ml of complete DMEM with 80 μM chloroquineis added to each flask and allowed to incubate an additional 3 hours.The medium is then aspirated and 24 ml of complete medium containing 10%DMSO for exactly 2 minutes and then aspirated. The cells are then washed2 times with PBS and 30 ml of complete DMEM added to each flask. Thecells are then allowed to incubate over night. The next day the cellsare harvested by trypsinization and reseeded as needed depending uponthe type of assay to be performed.

A typical protocol for liposomal-mediated transfection as applied to CHOcells is described as follows; Cells to be used for transfection aresplit 24 hours prior to the transfection to provide flasks which are70-80% confluent at the time of transfection. A total of 10 μg of DNAwhich may include varying ratios of receptor DNA plus any additional DNAneeded (e.g. G_(α) protein expression vector, reporter construct,antibiotic resistance marker, mock vector, etc.) is used to transfecteach 75 cm² flask of cells. Liposomal mediated transfection is carriedout according to the manufacturer's recommendations (LipofectAMINE,GibcoBRL, Bethesda, Md.). Transfected cells are harvested 24 h posttransfection and used or reseeded according the requirements of theassay to be employed.

A typical protocol for the electroporation method as applied to Cos-7cells is described as follows; Cells to be used for transfection aresplit 24 hours prior to the transfection to provide flasks which aresubconfluent at the time of transfection. The cells are harvested bytrypsinization resuspended in their growth media and counted. 4×10⁶cells are suspended in 300 μl of DMEM and placed into an electroporationcuvette. 8 μg of receptor DNA plus 8 μg of any additional DNA needed(e.g. G_(α) protein expression vector, reporter construct, antibioticresistance marker, mock vector, etc.) is added to the cell suspension,the cuvette is placed into a BioRad Gene Pulser and subjected to anelectrical pulse (Gene Pulser settings: 0.25 kV voltage, 950 μFcapacitance). Following the pulse, 800 μl of complete DMEM is added toeach cuvette and the suspension transferred to a sterile tube. Completemedium is added to each tube to bring the final cell concentration to1×10⁵ cells/100 μl. The cells are then plated as needed depending uponthe type of assay to be performed.

A typical protocol for viral mediated expression of heterolgous proteinsis described as follows for baculovirus infection of insect Sf9 cells.The coding region of DNA encoding the receptor disclosed herein may besubcloned into pBlueBacIII into existing restriction sites or sitesengineered into sequences 5′ and 3′ to the coding region of thepolypeptides. To generate baculovirus, 0.5 μg of viral DNA (BaculoGold)and 3 μg of DNA construct encoding a polypeptide may be co-transfectedinto 2×10⁶ Spodoptera frugiperda insect Sf9 cells by the calciumphosphate co-precipitation method, as outlined in by Pharmingen (in“Baculovirus Expression Vector System: Procedures and Methods Manual”).The cells then are incubated for 5 days at 27° C. The supernatant of theco-transfection plate may be collected by centrifugation and therecombinant virus plaque purified. The procedure to infect cells withvirus, to prepare stocks of virus and to titer the virus stocks are asdescribed in Pharmingen's manual. Similar principals would in generalapply to mammalian cell expression via retro-viruses, Simliki forestvirus and double stranded DNA viruses such as adeno-, herpes-, andvacinia-viruses, and the like.

Microinjection of cRNA encoding for proteins of interest is useful forthe study of protein function in xenopus oocytes as well as culturedmammalian cells. A typical protocol for the preparation of cRNA andinjection into xenopus oocytes can be found in the followingelectrophysiology section.

Stable Expression

Heterologous DNA can be stably incorporated into host cells, causing thecell to perpetually express a foreign protein. Methods for the deliveryof the DNA into the cell are similar to those described above fortransient expression but require the co-transfection of an ancillarygene to confer drug resistance on the targeted host cell. The ensuingdrug resistance can be exploited to select and maintain cells that havetaken up the heterologous DNA. An assortment of resistance genes areavailable including but not restricted to Neomycin, Kanamycin, andHygromycin. For the purposes of receptor studies, stable expression of aheterologous receptor protein is carried out in, but not necessarilyrestricted to, mammalian cells including, CHO, HEK293, LM(tk-), etc.

Cell Membrane Preparation

For binding assays, pellets of transfected cells are suspended inice-cold buffer (20 mM Tris.HCl, 5 mM EDTA, pH 7.4) and homogenized bysonication for 7 sec. The cell lysates are centrifuged at 200×g for 5min at 4° C. The supernatants are then centrifuged at 40,000×g for 20min at 4° C. The resulting pellets are washed once in the homogenizationbuffer and suspended in binding buffer (see methods for radioligandbinding). Protein concentrations are determined by the method ofBradford (1976) using bovine serum albumin as the standard. Bindingassays are usually performed immediately, however it is possible toprepare membranes in batch and store frozen in liquid nitrogen forfuture use.

Radioligand Binding Assays

Cells may be screened for the presence of endogenous human receptor byradioligand binding (described in detail below). Cells with either no ora low level of the endogenous human receptor disclosed herein may betransfected with the exogenous receptor. MCH1 binding experiments withmembranes (20-40 μg membrane protein) from transfected cells areperformed with 0.1 nM [¹²⁵I]Phe¹³-Tyr¹⁹-MCH (Custom labeled by NEN)using incubation buffer consisting of 50 mM Tris pH 7.4, 10 mM MgCl₂, 2μg/ml aprotonin, 0.5 mM PMSF and 50 μg/ml bacitracin. Binding isperformed at 25° C. for 1 hr. Incubations are terminated by rapid vacuumfiltration over GF/C glass fiber filters, presoaked in 5% PEI using 50mM Tris pH 7.4 containing 0.01% triton X-100 as wash buffer. In allexperiments nonspecific binding is defined using 10 μM unlabeled MCH.

Functional Assays

Cells may be screened for the presence of endogenous mammalian receptorusing functional assays (described in detail below). Cells with no or alow level of endogenous receptor present may be transfected with theexogenous receptor for use in the following functional assays.

A wide spectrum of assays can be employed to screen for receptoractivation. These range from traditional measurements of phosphatidylinositol, cAMP, Ca⁺⁺, and K⁺, for example; to systems measuring thesesame second messengers but which have been modified or adapted to behigher throughput, more generic, and more sensitive; to cell basedplatforms reporting more general cellular events resulting from receptoractivation such as metabolic changes, differentiation, and celldivision/proliferation, for example; to high level organism assays whichmonitor complex physiological or behavioral changes thought to beinvolved with receptor activation including cardiovascular, analgesic,orexigenic, anxiolytic, and sedation effects, for example.

Cyclic AMP (cAMP) Assay

The receptor-mediated stimulation or inhibition of cyclic AMP (cAMP)formation may be assayed in cells expressing the mammalian receptors.Cells are plated in 96-well plates and incubated in Dulbecco's phosphatebuffered saline (PBS) supplemented with 10 mM HEPES, 1 mMisobutylmethylxanthine for 20 min at 37° C., in 5% CON. Test compoundsare added with or without 10 μM forskolin and incubated for anadditional 10 min at 37° C. The medium is then aspirated and thereaction stopped by the addition of 100 mM HCl. The plates are stored at4° C. for 15 min, and the cAMP content in the stopping solution measuredby radioimmunoassay. Radioactivity may be quantified using a gammacounter equipped with data reduction software.

Arachidonic Acid Release Assay

Cells expressing the mammalian receptor are seeded into 96 well platesand grown for 3 days in HAM's F-12 with supplements. [³H]-arachidonicacid (specific activity=0.75 μCi/ml) is delivered as a 100 μL aliquot toeach well and samples were incubated at 37° C., 50% CO₂ for 18 hours.The labeled cells are washed three times with 200 μL HAM's F-12. Thewells are then filled with medium (200 μL) and the assay is initiatedwith the addition of peptides or buffer (22 μL). Cells are incubated for30 mim at 37° C., 5% CO₂. Supernatants are transferred to a microtiterplate and evaporated to dryness at 75° C. in a vacuum oven. Samples arethen dissolved and resuspended in 25 μL distilled water. Scintillant(300 μL) is added to each well and samples are counted for ³H in aTrilux plate reader. Data are analyzed using nonlinear regression andstatistical techniques available in the GraphPAD Prism package (SanDiego, Calif.).

Intracellular Calcium Mobilization Assay

The intracellular free calcium concentration may be measured bymicrospectroflourometry using the fluorescent indicator dye Fura-2/AM(Bush et al, 1991). Cells are seeded onto a 35 mm culture dishcontaining a glass coverslip insert, washed with HBS and loaded with 100μL of Fura-2/AM (10 μM) for 20 to 40 min. After washing with HBS toremove the Fura-2/AM solution, cells are equilibrated in HBS for 10 to20 min. Cells are then visualized under the 40× objective of a LeitzFluovert FS microscope and fluorescence emission is determined at 510 nMwith excitation wavelengths alternating between 340 nM and 380 nM. Rawfluorescence data are converted to calcium concentrations using standardcalcium concentration curves and software analysis techniques.

Inositol Phosphate Assay

Guidelines for cell preparation and assay of the second messengerinositol phosphate (IP) are described below for a typical protocolinvolving transiently transfected Cos-7 cells; For a 96 well microplateformat assay, cells are plated at 70,000 cells per well and allowed toincubate for 24 hours after the transfection procedure. The cells arethen labeled with 0.5 μCi [³H]myo-inositol per micro-well over night at37° C., 5% CO₂. Immediately before the assay, the medium is removed andreplaced with 90 μl PBS containing 10 mM LiCl. The plates are thenincubated for 15 minutes at 37° C., 5% CO₂. Following the incubation,the transfectants are challenged with agonist (10 μl/well; 10×concentration) for 30 minutes at 37° C., 5% CO₂. The challenge isterminated and the cells lysed by the addition of 100 μu cold 5% v/vtrichloroacetic acid (TCA), followed by an incubation at 4° C. forgreater than 30 minutes. Total IPs are isolated from the lysate by ionexchange chromatography. Briefly, the lysed contents of the wells aretransferred to a Multiscreen HV filter plate (Millipore) containing 100μl Dowex AG1-X8 suspension (50% v/v, water:resin) (200-400 mesh, formateform). The filter plates are placed on a vacuum manifold to wash andelute the resin bed. Each well is first washed 2 times with 200 μl 5 mMmyoinositol. Total [³H]IPs are eluted with 75 μl of 1.2 M ammoniumformate/0.1 M formic acid into Wallac 96-well plates. 200 μl of SuperMixscintillation cocktail is added to each well, mixed well, allowed toequilibrate and counted on a Micro Beta Trilux scintillation counter.(Note: The assay may be scaled to a 24 well format by simple adjustmentof reagent volumes and employing individual chromatographic columns.)

GTPγS Functional Assay

Membranes from cells transfected with the mammalian receptors aresuspended in assay buffer (50 mM Tris, 100 mM NaCl, 5 mM MgCl₂, pH 7.4)supplemented with 0.2% BSA and 10 μM GDP. Membranes are incubated on icefor 20 minutes, transferred to a 96-well Millipore microtiter GF/Cfilter plate and mixed with GTPγ³⁵S (e.g., 250,000 cpm/sample, specificactivity ˜1000 Ci/mmol) plus or minus GTPγS (final concentration=100μM). Final membrane protein concentration≈90 μg/ml. Samples areincubated in the presence or absence of MCH (final concentration=1 μM)for 30 min. at room temperature, then filtered on a Millipore vacuummanifold and washed three times with cold assay buffer. Samplescollected in the filter plate are treated with scintillant and countedfor ³⁵S in a Trilux (Wallac) liquid scintillation counter. It isexpected that optimal results are obtained when the mammalian receptormembrane preparation is derived from an appropriately engineeredheterologous expression system, i.e., an expression system resulting inhigh levels of expression of the mammalian receptor and/or expressingG-proteins having high turnover rates (for the exchange of GDP for GTP).GTPγS assays are well-known in the art, and it is expected thatvariations on the method described above, such as are described by e.g.,Tian et al. (1994) or Lazareno and Birdsall (1993), may be used by oneof ordinary skill in the art.

Transcription Assay

Guidelines for cell preparation and assay of receptor mediatedtranscription of Cos-7 cells transiently transfected by the DEAE-dextranmethod in a 96 microwell format is as follows; The c-fos-β-galpromoter/reporter construct used for these studies consists of the cfospromoter region (−384 to +19) (Schilling et al 1991, Yalkinoglu et al,1995) inserted upstream of β-galactosidase cDNA containing expressionvector pNASSβ (Clontech). Transcription activity is measured by assay ofβ-galactosidase enzyme activity as detected in a calorimetric assay.Forty-eight hours following transient transfection, the medium isremoved and replaced with medium containing drug (e.g. MCH) typically ata concentration of 10 μM. The cells are allowed to incubate at 37° C.,5% CO₂ for at least 18 hours, after which the medium is aspirated andthe cells washed with 200 μl PBS/well. The cells are then lysed with 100μl AB buffer (100 mM Sodium Phosphate buffer, pH 8.0, 2 mM MgSO₄, 0.1 mMMnCl₂) for 10 minutes at room temperature. 100 μl ofAB/Tx/β-mercaptoethanol (AB buffer with 0.5% Triton X-100, 40 mMβ-mercaptoethanol) is then added to each well and the lysate allowed toincubate an additional 10 minutes at room temperature. The enzymaticcolor reaction is initiated by the addition of the substrate, ONPG/AB (4mg/ml O-nitrophenyl-b-D-galactopyranoside in AB buffer). The reaction isallowed to proceed for 30 minutes or until yellow color becomes evident.Measurement of optical density is taken at 405 nm using a Dynatechmicroplate reader.

MAP Kinase Assay

MAP kinase (mitogen activated kinase) may be monitored to evaluatereceptor activation. MAP kinase is activated by multiple pathways in thecell. A primary mode of activation involves the ras/raf/MEK/MAP kinasepathway. Growth factor (tyrosine kinase) receptors feed into thispathway via SHC/Grb-2/SOS/ras. Gi coupled receptors are also known toactivate ras and subsequently produce an activation of MAP kinase.Receptors that activate phospholipase C (Gq and G11) producediacylglycerol (DAG) as a consequence of phosphatidyl inositolhydrolysis. DAG activates protein kinase C which in turn phosphorylatesMAP kinase.

MAP kinase activation can be detected by several approaches. Oneapproach is based on an evaluation of the phosphorylation state, eitherunphosphorylated (inactive) or phosphorylated (active). Thephosphorylated protein has a slower mobility in SDS-PAGE and cantherefore be compared with the unstimulated protein using Westernblotting. Alternatively, antibodies specific for the phosphorylatedprotein are available (New England Biolabs) which can be used to detectan increase in the phosphorylated kinase. In either method, cells arestimulated with the mitogen and then extracted with Laemmli buffer. Thesoluble fraction is applied to an SDS-PAGE gel and proteins aretransferred electrophoretically to nitrocellulose or Immobilon.Immunoreactive bands are detected by standard Western blottingtechnique. Visible or chemiluminescent signals are recorded on film andmay be quantified by densitometry.

Another approach is based on evaluation of the MAP kinase activity via aphosphorylation assay. Cells are stimulated with the mitogen and asoluble extract is prepared. The extract is incubated at 30° C. for 10min with gamma-32-ATP, an ATP regenerating system, and a specificsubstrate for MAP kinase such as phosphorylated heat and acid stableprotein regulated by insulin, or PHAS-I. The reaction is terminated bythe addition of H₃PO₄ and samples are transferred to ice. An aliquot isspotted onto Whatman P81 chromatography paper, which retains thephosphorylated protein. The chromatography paper is washed and countedfor ³²P in a liquid scintillation counter. Alternatively, the cellextract is incubated with gamma-32-ATP, an ATP regenerating system, andbiotinylated myelin basic protein bound by streptavidin to a filtersupport. The myelin basic protein is a substrate for activated MAPkinase. The phosphorylation reaction is carried out for 10 min at 30° C.The extract can then be aspirated through the filter, which retains thephosphorylated myelin basic protein. The filter is washed and countedfor ³²P by liquid scintillation counting.

Cell Proliferation Assay

Activation of a G protein coupled receptor may lead to a mitogenic orproliferative response which can be monitored via [³H]-thymidine uptake.When cultured cells are incubated with [³H]-thymidine, the thymidinetranslocates into the nuclei where it is phosphorylated to thymidinetriphosphate. The nucleotide triphosphate is then incorporated into thecellular DNA at a rate that is proportional to the rate of cell growth.Typically, cells are grown in culture for 1-3 days. Cells are forcedinto quiescence by the removal of serum for 24 hrs. A mitogenic agent isthen added to the media. 24 hrs later, the cells are incubated with[H]-thymidine at specific activities ranging from 1 to 10 μCi/ml for 2-6hrs. Harvesting procedures may involve trypsinization and trapping ofcells by filtration over GF/C filters with or without a prior incubationin TCA to extract soluble thymidine. The filters are processed withscintillant and counted for ³H by liquid scintillation counting.Alternatively, adherent cells are fixed in MeOH or TCA, washed in water,and solubilized in 0.05% deoxycholate/0.1 N NaOH. The soluble extract istransferred to scintillation vials and counted for ³H by liquidscintillation counting.

Methods for Recording Currents in Xenopus Oocytes

Female Xenopus laevis (Xenopus-1, Ann Arbor, Mich.) are anesthetized in0.2% tricain (3-aminobenzoic acid ethyl ester, Sigma Chemical Corp.) anda portion of ovary is removed using aseptic technique (Quick and Lester,1994). Oocytes are defolliculated using 2 mg/ml collagenase (WorthingtonBiochemical Corp., Freehold, N.J.) in a solution containing 87.5 mMNaCl, 2 mM KCl, 2 mM MgCl₂ and 5 mM HEPES, pH 7.5. Oocytes may beinjected (Nanoject, Drummond Scientific, Broomall, Pa.) with mammalianmRNA. Other oocytes may be injected with a mixture of mammalian mRNA andmRNA encoding the genes for G-protein-activated inward rectifiers (GIRK1and GIRK4, U.S. Pat. Nos. 5,734,021 and 5,728,535). Genes encodingG-protein inwardly rectifying K⁺ (GIRK) channels 1 and 4 (GIRK1 andGIRK4) were obtained by PCR using the published sequences (Kubo et al.,1993; Dascal et al., 1993; Krapivinsky et al., 1995 and 1995b) to deriveappropriate 5′ and 3′ primers. Human heart cDNA was used as templatetogether with the primers

5′-CGCGGATCCATTATGTCTGCACTCCGAAGGAAATTTG-3′ (Seq. I.D. No. 10) and

5′-CGCGAATTCTTATGTGAAGCGATCAGAGTTCATTTTTC-3′ (Seq. I.D. No. 11) forGIRK1 and

5′-GCGGGATCCGCTATGGCTGGTGATTCTAGGAATG-3′ (Seq. I.D. No. 12) and

5′- CCGGAATTCCCCTCACACCGAGCCCCTGG-3′ (Seq. I.D. No. 13) for GIRK4.

In each primer pair, the upstream primer contained a BamHI site and thedownstream primer contained an EcoRI site to facilitate cloning of thePCR product into pcDNA1-Amp (Invitrogen). The transcription template forthe mammalian receptor may be similarly obtained. mRNAs are preparedfrom separate DNA plasmids containing the complete coding regions of themammalian receptor, GIRK1, and GIRK4. Plasmids are linearized andtranscribed using the T7 polymerase (“Message Machine”, Ambion).Alternatively, mRNA may be translated from a template generated by PCR,incorporating a T7 promoter and a poly A⁺ tail. Each oocyte receives 2ng each of GIRK1 and GIRK4 mRNA in combination with 25 ng of mammalianreceptor mRNA. After injection of mRNA, oocytes are incubated at 16° C.on a rotating platform for 3-8 days. Dual electrode voltage clamp(“GeneClamp”, Axon Instruments Inc., Foster City, Calif.) is performedusing 3 M KCl-filled glass microelectrodes having resistances of 1-3Mohms. Unless otherwise specified, oocytes are voltage clamped at aholding potential of −80 mV. During recordings, oocytes are bathed incontinuously flowing (2-5 ml/min) medium containing 96 mM NaCl, 2 mMKCl, 2 mM CaCl₂, 2 mM MgCl₂, and 5 mM HEPES, pH 7.5 (“ND96”), or, in thecase of oocytes expressing GIRK1 and GIRK4, elevated K⁺ containing 96 mMKCl, 2 mM NaCl, 2 mM CaCl₂, 2 mM MgCl, and 5 mM HEPES, pH 7.5 (“hK”).Drugs are applied by switching from a series of gravity fed perfusionlines.

Heterologous expression of GPCRs in Xenopus oocytes has been widely usedto determine the identity of signaling pathways activated by agoniststimulation (Gundersen et al., 1983; Takahashi et al., 1987). Activationof the phospholipase C (PLC) pathway is assayed by applying testcompound in ND96 solution to oocytes previously injected with mRNA forthe mammalian receptor and observing inward currents at a holdingpotential of −80 mV. The appearance of currents that reverse at −25 mVand display other properties of the Ca⁺⁺-activated Cl⁻ (chloride)channel is indicative of mammalian receptor-activation of PLC andrelease of IP3 and intracellular Ca⁺⁺. Such activity is exhibited byGPCRs that couple to G_(q).

Measurement of inwardly rectifying K⁺ (potassium) channel (GIRK)activity is monitored in oocytes that have been co-injected with mRNAsencoding the mammalian receptor, GIRK1, and GIRK4. The two GIRK geneproducts co-assemble to form a G-protein activated potassium channelknown to be activated (i.e., stimulated) by a number of GPCRs thatcouple to G_(i) or G_(o) (Kubo et al., 1993; Dascal et al., 1993).Oocytes expressing the mammalian receptor plus the two GIRK subunits aretested for test compound responsivity by measuring K⁺ currents inelevated K⁺ solution (hK). Activation of inwardly rectifying currentsthat are sensitive to 300 μM Ba⁺⁺ signifies the mammalian receptorcoupling to a G_(i) or G_(o) pathway in the oocytes.

Receptor/G Protein Co-Transfection Studies

A strategy for determining whether MCH1 can couple preferentially toselected G proteins involves co-transfection of MCH1 receptor cDNA intoa host cell together with the cDNA for a G protein alpha sub-unit.Examples of G alpha sub-units include members of the Gαi/Gαo class(including Gαt2 and Gαz), the Gαq class, the Gαs class, and the Gα12/13class. A typical procedure involves transient transfection into a hostcell such as COS-7. Other host cells may be used. A key consideration iswhether the cell has a downstream effector (a particular adenylatecyclase, phospholipase C, or channel isoform, for example) to support afunctional response through the G protein under investigation. G proteinbeta gamma sub-units native to the cell are presumed to complete the Gprotein heterotrimer; otherwise specific beta and gamma sub-units may beco-transfected as well. Additionally, any individual or combination ofalpha, beta, or gamma subunits may be co-transfected to optimize thefunctional signal mediated by the receptor.

The receptor/G alpha co-transfected cells are evaluated in a bindingassay, in which case the radioligand binding may be enhanced by thepresence of the optimal G protein coupling or in a functional assaydesigned to test the receptor/G protein hypothesis. In one example, theMCH1 receptor may be hypothesized to inhibit cAMP accumulation throughcoupling with G alpha sub-units of the Gαi/Gαo class. Host cellsco-transfected with the MCH1 receptor and appropriate G alpha sub-unitcDNA are stimulated with forskolin +/− MCH1 agonist, as described abovein cAMP methods. Intracellular cAMP is extracted for analysis byradioimmunoassay. Other assays may be substituted for cAMP inhibition,including GTPγ³⁵S binding assays and inositol phosphate hydrolysisassays. Host cells transfected with MCH1 minus G alpha or with G alphaminus MCH1 would be tested simultaneously as negative controls. MCH1receptor expression in transfected cells may be confirmed in radioligandbinding studies using membranes from transfected cells. G alphaexpression in transfected cells may be confirmed by Western blotanalysis of membranes from transfected cells, using antibodies specificfor the G protein of interest.

The efficiency of the transient transfection procedure is a criticalfactor for signal to noise in an inhibitory assay, much more so than ina stimulatory assay. If a positive signal present in all cells (such asforskolin-stimulated cAMP accumulation) is inhibited only in thefraction of cells successfully transfected with receptor and G alpha,the signal to noise ratio will be poor. One method for improving thesignal to noise ratio is to create a stably transfected cell line inwhich 100% of the cells express both the receptor and the G alphasubunit. Another method involves transient co-transfection with a thirdcDNA for a G protein-coupled receptor which positively regulates thesignal which is to be inhibited. If the co-transfected cellssimultaneously express the stimulatory receptor, the inhibitoryreceptor, and a requisite G protein for the inhibitory receptor, then apositive signal may be elevated selectively in transfected cells using areceptor-specific agonist. An example involves co-transfection of COS-7cells with 5-HT4 receptor, MCH1 receptor, and a G alpha sub-unit.Transfected cells are stimulated with a 5-HT4 agonist +/− MCH1 agonist.Cyclic AMP is expected to be elevated only in the cells also expressingMCH1 and the G alpha subunit of interest, and a MCH1-dependentinhibition may be measured with an improved signal to noise ratio.

It is to be understood that the cell lines described herein are merelyillustrative of the methods used to evaluate the binding and function ofthe mammalian receptors of the present invention, and that othersuitable cells may be used in the assays described herein.

Promiscuous Second Messenger Assays

It is possible to coax receptors of different functional classes tosignal through a pre-selected pathway through the use of promiscuousG_(α) subunits. For example, by providing a cell based receptor assaysystem with an exogenously supplied promiscuous G_(α), subunit such asG_(α16) or a chimeric G_(α) subunit such as G_(αzq), a GPCR whichnormally might prefer to couple through a specific signaling pathway(e.g. G_(s), G_(i), G_(q), G_(o), etc.), can be made to couple throughthe pathway defined by the promiscuous G_(α) subunit and upon agonistactivation produce the second messenger associated with that subunit'spathway. In the case of G_(α16) and/or G_(αqz) this would involveactivation of the G_(q) pathway and production of the second messengerinositol phosphate. Through similar strategies and tools, it is possibleto bias receptor signaling through pathways producing other secondmessengers such as Ca⁺⁺, cAMP, K⁺ currents, etc.

Microphysiometric Assay

Because cellular metabolism is intricately involved in and effected by abroad range of cellular events (including receptor activation of varioussecond messenger pathways), the use of microphysiometric measurements ofcell metabolism can in principle provide a generic assay of cellularactivity arising from the activation of any receptor regardless of thespecifics of the receptor's proximal signaling pathway. Generalguidelines for cell preparation and microphysiometric recording havebeen previously reported (Salon, J. A. and Owicki, J. A., 1996). Atypical protocol employing transiently transfected CHO cells is asfollows; 24 hours prior to recording, transfected cells are harvestedand counted. 3×10⁵ cells are seeded into cell culture capsules (Costar),and allowed to attach to the capsule membrane. 10 hours later (14 hoursprior to recording) the cell media is switched to serum free F-12complete to minimize ill-defined metabolic stimulation caused byassorted serum factors.

On the day of the experiment the cell capsules are transferred to themicrophysiometer (Cytosensor, Molecular Devices Corporation, Sunnyvale,Calif.) and allowed to equilibrate in recording media (low buffered RPMI1640, no bicarbonate, no serum) with 0.1% BSA (essentially fatty acidfree), during which a baseline measurement of basal metabolic activityis established. The recording paradigm consists of a 100 μl/min flowrate, with a 2 min pump cycle which includes a 30 sec flow interruptionduring which the rate measurement is taken. Challenges involve a 1 min20 sec exposure to a drug just prior to the first post challenge ratemeasurement being taken, followed by two additional pump cycles for atotal of 5 min 20 sec drug exposure. Drug is then washed out and ratesallowed to return to basal. Reported extracellular acidification ratesare expressed as a percentage increase of the peak response over thebaseline rate observed just prior to challenge.

GPCR Ligand Library

Functional assays of new receptors such as MCH1 may include apreliminary test of a small library of compounds containingrepresentative agonists for all known GPCRs as well as other compoundswhich may be agonists for prospective GPCRs or which may be effectorsfor targets peripherally involved with GPCRs. The collection used inthis study comprises approximately 180 compounds (including smallmolecules, hormones, preprohormones, peptides, etc.) for more than 45described classes of GPCRs (serotonin, dopamine, noradrenaline, opioids,etc.) and additionally includes ligands for known or suspected but notnecessarily pharmacological characterized or cloned GPCR families (suchas MCH).

The diversity of the library can be expanded to include agonist andantagonist compounds specific for GPCR subtypes, combinatorial peptideand/or small molecule libraries, natural product collections, and thelike. To facilitate robotic handling, the substances are distributed aseither separate or pooled compound concentrates in 96 well plates andstored frozen as ready to use reagent plates.

Localization of mRNA Coding For Human MCH1 Receptors

Development of probes for MCH1: To facilitate the production ofradiolabeled, antisense RNA probes a fragment of the gene encoding ratMCH1 will be subcloned into a plasmid vector containing RNA polymerasepromoter sites. The full length cDNA encoding the rat MCH1 will bedigested with Pst 1, (nucleotides 905-1194) and this 289 nucleotidefragment will be cloned into the Pst I site of PGEM 3z, containing bothsp6 and T7 RNA polymerase promoter sites. The construct will besequenced to confirm sequence identity and orientation. To synthesizeantisense strands of RNA, this construct will be linearized with HindIII or Eco RI (depending on orientation) and T7 or sp6 RNA polymerasewill be used to incorporate radiolabeled nucleotide as described below.

A probe coding for the rat glyceraldehyde 3-phosphate dehydrogenase(GAPDH) gene, a constitutively expressed protein, was used concurrently.GAPDH is expressed at a relatively constant level in most tissue and itsdetection is used to compare expression levels of the rat MCH1 receptorsgene in different regions. Synthesis of probes: MCH1 and GAPDH cDNAsequences preceded by phage polymerase promoter sequences will be usedto synthesize radiolabeled riboprobes. Conditions for the synthesis ofriboprobes will be: 0.25-1.0 μg linearized DNA plasmid template, 1.5 μlof ATP, GTP, UTP (10 mM each), 3 μl dithiothreitol (0.1 M), 30 unitsRNAsin RNAse inhibitor, 0.5-1.0 μl (15-20 units/μl) RNA polymerase, 7.0μl transcription buffer (Promega Corp.), and 12.5 μl α³²P-CTP (specificactivity 3,000 Ci/mmol). 0.1 mM CTP (0.02-1.0 μl) will be added to thereactions, and the volume will be adjusted to 35 μl with DEPC-treatedwater. Labeling reactions will be incubated at 37° C. for 60 min, afterwhich 3 units of RQ1 RNAse-free DNAse (Promega Corp.) will be added todigest the template. Riboprobes will be separated from unincorporatednucleotides using Microspin S-300 columns (Pharmacia Biotech). TCAprecipitation and liquid scintillation spectrometry will be used tomeasure the amount of label incorporated into the probe. A fraction ofall riboprobes synthesized will be size-fractionated on 0.25 mm thick 7Murea, 4.5% acrylamide sequencing gels. These gels will be apposed tostorage phosphor screens and the resulting autoradiograph scanned usinga phoshorimager (Molecular Dynamics, Sunnyvale, Calif.) to confirm thatthe probes synthesized were full-length and not degraded.

Solution Hybridization/Ribonuclease Protection Assay

(RPA): For solution hybridization 2.0 μg of mRNA isolated from tissueswill be used. Negative controls consisted of 30 μg transfer RNA (tRNA)or no tissue blanks. All mRNA samples will be placed in 1.5-ml microfugetubes and vacuum dried. Hybridization buffer (40 μl of 400 mM NaCl, 20mM Tris, pH 6.4, 2 mM EDTA, in 80% formamide) containing 0.25-2.0E⁶counts of each probe will be added to each tube. Samples will beheated at 95° C. for 15 min, after which the temperature will be loweredto 55° C. for hybridization.

After hybridization for 14-18 hr, the RNA/probe mixtures will bedigested with RNAse A (Sigma) and RNAse T1 (Life Technologies). Amixture of 2.0 μg RNAse A and 1000 units of RNAse T1 in a buffercontaining 330 mM NaCl, 10 mM Tris (pH 8.0) and 5 mM EDTA (400 μl) willbe added to each sample and incubated for 90 min at room temperature.After digestion with RNAses, 20 μl of 10% SDS and 50 μg proteinase Kwill be added to each tube and incubated at 37° C. for 15 min. Sampleswill be extracted with phenol/chloroform:isoamyl alcohol andprecipitated in 2 volumes of ethanol for 1 hr at −70° C. Pellet Paint(Novagen) will be added to each tube (2.0 μg) as a carrier to facilitateprecipitation. Following precipitation, samples will be centrifuged,washed with cold 70% ethanol, and vacuum dried. Samples will bedissolved in formamide loading buffer and size-fractionated on aurea/acrylamide sequencing gel (7.0 M urea, 4.5% acrylamide inTris-borate-EDTA). Gels will be dried and apposed to storage phosphorscreens and scanned using a phosphorimager (Molecular Dynamics,Sunnyvale, Calif.).

RT-PCR: For the detection of RNA encoding human MCH1, RT-PCR was carriedout on mRNA extracted from human tissue. Reverse transcription and PCRreactions were carried out in 50 ml volumes using EZrTth DNA polymerase(Perkin Elmer). Primers with the following sequences were used:

Forward primer (RA SLCla/MCH F); TCA GCT CGG TTG TGG GAG CA (Seq. I.D.No. 14)

Reverse primer (RA/SLCla MCH B); CTT GGA CTT CTT CAC GAC (Seq. I.D. No.15)

These primers will amplify a 248 base pair fragment from nucleotide 169to 417.

Each reaction contained 0.1 μg mRNA and 0.3μM of each primer.Concentrations of reagents in each reaction were: 300 μM each of GTP;DATP; dCTP; dTTP; 2.5 mM Mn(OAc)2; 50 mM Bicine; 115 mM potassiumacetate, 8% glycerol and 5 units EZrTth DNA polymerase. All reagents forPCR (except mRNA and oligonucleotide primers) were obtained from PerkinElmer. Reactions were carried out under the following conditions: 65° C.60 min., 94° C. 2 min., (94° C., 1 min., 65° C. 1 min) 35 cycles, 72° C.10 min. PCR reactions were size fractionated by gel electrophoresisusing 10% polyacrylamide. DNA was stained with SYBR Green I (MolecularProbes, Eugene Oreg.) and scanned on a Molecular Dynamics (SunnyvaleCalif.) Storm 860 in blue fluorescence mode at 450 nM.

Positive controls for PCR reactions consisted of amplification of thetarget sequence from a plasmid construct, as well as reversetranscribing and amplifying a known sequence. Negative controlsconsisted of mRNA blanks, as well as primer and mRNA blanks. To confirmthat the mRNA was not contaminated with genomic DNA, samples weredigested with RNAses before reverse transcription. Integrity of RNA wasassessed by amplification of mRNA coding for GAPDH.

Results and Discussion

Cloning and Sequencing

Discovery of an Expressed Sequence Tag (EST) F07228 in GENEML Homologousto FB41a

A BLAST search of GENEMBL with a Synaptic Pharmaceutical Corporationproprietary sequence, FB41a, resulted in the identification of an EST(accession number F07228) with a high degree of homology to FB41a andsomatostatin, opiate and galanin receptors.

Construction and Screening of a Human Hippocampal cDNA Library

A human hippocampal cDNA library containing a total of 2.2×10⁶independent clones with a mean insert size of 3.0 kb was prepared in theexpression vector PEXJ.BS. The library was plated on agar plates(ampicillin selection) and glycerol stocks for 450 pools of 5000independent clones were prepared. Primary glycerol stocks were alsogrouped together in groups of approximately 10 to create superpools.

Cloning of the Full-Length Sequence of MCH1

Glycerol stocks of the superpools and primary pools from the humanhippocampal cDNA library were screened by PCR with F07228 specificprimers T579 and T580. One positive primary pool 490, was successivelydivided into subpools, amplified in LB medium overnight and screened byPCR using primers T579 and T580. One positive subpool, 490-4-10-23 wasplated on agar plates (ampicillin selection), and colonies weretransferred to nitrocellulose membranes (Schleicher and Schuell, Keene,NH). Filters were hybridized for two days under high stringencyconditions with 10⁶ cpm/ml of a ³²P-labeled cDNA probe, T581, designedagainst the F07228 EST sequence. Filters were washed and apposed toBiomax MS film (Kodak). Seven positive colonies were picked, streaked onLB-AMP plates, and grown overnight. Two individual colonies from each ofthe original seven were picked and subjected to vector-anchored PCRusing the following primer pairs: T95, T580 and T94, T579. One positivecolony, G1, was amplified overnight in TB and processed for plasmidpurification. This plasmid was designated TL230 and sequenced on bothstrands. Nucleotide and peptide sequence analysis were performed withGCG programs (Genetics Computer Group, Madison, Wis.). A HindIII-KpIfragment of TL230 was subcloned into the mammalian expression vectorpEXJ, and named TL231. The largest open reading frame in this constructcontains 1266 nucleotides (FIG. 1), which is predicted to encode aprotein of 422 amino acids (FIG. 2). There are three in-framemethionines in the amino terminus which could result in a protein of422, 417 or 353 amino acids. Hydropathy analysis of the protein isconsistent with a putative topography of seven transmembrane domains,indicative of the G protein-coupled receptor family (FIG. 3). TL231 hasbeen named MCH1.

Database analysis of the sequence of MCH1 revealed that it was mostsimilar to somatostatin receptors. Further database analysis revealed aGenbank submission (accession number AF008650, deposited on Oct. 1,1997) which appears to be the rat homologue of TL231. AF008650 is 69nucleotides shorter than MCH1 at the 5′ end, and predicts a differentinitiating methionine. FIGS. 4 and 5 illustrate the nucleotide and aminoacid sequence for the rat MCH1 receptor, respectively.

Inositol Phosphate Response of MCH1-Transfected Cells

The expression vector (pEXJ) containing the MCH1 cDNA was transfected byelectroporation into Cos-7 cells in combination with an expressionvector (pEXJ) containing the G_(α16) subunit. After plating and labelingwith [³H]-myo-inositol, the transfectants were challenged with a ligandlibrary that included, among other things, melanin concentrating hormone(MCH) (10 μM final concentration) and then assayed for inositolphosphate (IP) formation. In five out of the seven screens, cellstransfected with MCH1 (with G_(α16)) gave an approximately 1.4-foldincrease in IP production as compared to cells transfected with G_(α16)alone when challenged with MCH.

Subsequent experiments demonstrated that 10 μM MCH was able to stimulateIP release 3.4-fold over basal levels in Cos-7 cells transfected withMCH1 alone, suggesting that this receptor couples through the G_(q)signaling pathway. The IP response was shown to be dose-dependent to MCHwith an EC₅₀ value of 9.3±1.7 nM (n=2) and an E_(max) of approximately400% basal (404±72) (FIG. 6).

Several additional compounds were tested for their ability to activateMCH1. No dose-responsiveness of inositol phosphate formation could bedetected in Cos-7 cells transfected with MCH1 when challenged withsomatostatin, haloperidol, or dynorphin A1-13, discounting thepossibility that MCH1 encodes a somatostatin-like or opioid-like orsigma-like GPCR subtype (FIG. 7)

Microphysiometric Response of MCH1-Transfected Cells to MCH

CHO cells were transiently transfected with MCH1 using lipofectant,challenged with increasing concentrations of MCH or Phe¹³,Tyr¹⁹-MCH, andsubsequently monitored for changes in extracellular acidification rates.Both ligands produced a dose-dependent increase in acidification ratewith an EC₅₀ value of 8.6 nM for MCH and 51.8 nM for Phe¹³,Tyr¹⁹-MCH.Neither native CHO cells or mock (pEXJ) transfected CHO cells exhibiteda change in acidification rate when exposed to MCH or Phe¹³,Tyr¹⁹-MCH(FIG. 8).

Transcriptional Response of MCH1-Transfected Cells

Cos-7 cells were transiently transfected with MCH1 and a c-fos-β-galreporter construct by the DEAE-dextran method. The cells were challengedwith assorted drugs, including MCH, and transcriptional activitymeasured by calorimetric assay of β-galactosidase protein expression.Initial single dose challenges with MCH at a concentration of 10 μMstimulated c-fos-regulated transcriptional activity approximately3.9-fold over cells challenged with medium only. Cells transfected withonly the c-fos-β-gal construct showed no response to MCH. Subsequentexperimentation showed the transcription activation response to bedose-dependent to MCH with an EC₅₀ value of 116 nM (FIG. 9).

Binding of [¹²⁵I]Phe¹³,Tyr¹⁹-MCH in MCH1-Transfected Cells

Membranes harvested from Cos-7 cells transfected with MCH1 by theDEAE-dextran method exhibited specific binding for [¹²⁵I]Phe¹³-Tyr¹⁹-MCH(about 80 fmol/mg membrane protein) over mock-transfected cells (about20 fmol/mg membrane protein) at 0.1 nM radioligand concentration.Specific [¹²⁵I]Phe¹³Tyr¹⁹-MCH binding was about 70% of total binding ata radioligand concentration of 0.1 nM (FIG. 10).

Localization of mRNA Encoding Human MCH1 Receptors

RT-PCR was used to assess the presence of MCH1 receptor encoding messagein mRNA samples isolated from a variety of human tissues (Table 1, FIG.11). After amplification, PCR reactions were size fractionated on 10%polyacrylamide gels, and stained with SYBR Green I. Images were analyzedusing a Molecular Dynamics Storm 860 workstation. The amplified bandcorresponding to MCH1 receptor (490 base pairs) is indicated (arrow)RT-PCR analysis indicates the distribution of mRNA encoding human MCH1receptor is widespread throughout all tissues assayed, including bothcentral nervous system tissue and peripheral organs. This widespreaddistribution implies broad regulatory functions that involve nervoussystem as well as endocrine mechanisms.

TABLE 1 Distribution of mRNA coding for human MCH1 receptors. humanRegion MCH 1 Potential applications liver +++ Diabetes kidney +++Hypertension, Electrolyte balance lung +++ Respiratory disorders, asthmaheart +++ Cardiovascular indications small intestine +++Gastrointestinal disorders striated muscle +++ Musculoskeletal disorderspituitary +++ Endocrine/neuroendocrine regulation whole brain +++amygdala +++ Depression, phobias, anxiety, mood disorders cerebralcortex +++ Sensory and motor integration, cognition hippocampus +++Cognition/memory hypothalamus +++ appetite/obesity, neuroendocrineregulation spinal cord +++ Analgesia, sensory modulation andtransmission cerebellum +++ Motor coordination thalamus +++ sensoryintegration substantia +++ Modulation of dopaminergic nigra function.Modulation of motor coordination. caudate-putamen +++ Modulation ofdopaminergic function fetal brain +++ Developmental disorders fetal lung+++ Developmental disorders fetal kidney +++ Developmental disordersfetal liver +++ Developmental disorders

The cloning of the gene encoding the human MCH1 receptor has providedthe means to explore its physiological role by pharmacologicalcharacterization, and by Northern and in situ mapping of its mRNAdistribution. Further, the availability of the DNA encoding the humanMCH1 receptor will facilitate the development of antibodies andantisense technologies useful in defining the functions of the geneproducts in vivo. Antisense oligonucleotides which target mRNA moleculesto selectively block translation of the gene products in vivo have beenused successfully to relate the expression of a single gene with itsfunctional sequelae. Thus, the cloning of this receptor gene providesthe means to explore its physiological role in the nervous system andelsewhere, and may thereby help to elucidate structure/functionrelationships within the GPCR superfamily.

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15 1 1269 DNA Homo sapiens 1 atgtcagtgg gagccatgaa gaagggagtg gggagggcagttgggcttgg aggcggcagc 60 ggctgccagg ctacggagga agaccccctt cccgactgcggggcttgcgc tccgggacaa 120 ggtggcaggc gctggaggct gccgcagcct gcgtgggtggaggggagctc agctcggttg 180 tgggagcagg cgaccggcac tggctggatg gacctggaagcctcgctgct gcccactggt 240 cccaatgcca gcaacacctc tgatggcccc gataacctcacttcagcagg atcacctcct 300 cgcacgggga gcatctccta catcaacatc atcatgccttcggtgttcgg caccatctgc 360 ctcctgggca tcatcgggaa ctccacggtc atcttcgcggtcgtgaagaa gtccaagctg 420 cactggtgca acaacgtccc cgacatcttc atcatcaacctctcggtagt agatctcctc 480 tttctcctgg gcatgccctt catgatccac cagctcatgggcaatggggt gtggcacttt 540 ggggagacca tgtgcaccct catcacggcc atggatgccaatagtcagtt caccagcacc 600 tacatcctga ccgccatggc cattgaccgc tacctggccactgtccaccc catctcttcc 660 acgaagttcc ggaagccctc tgtggccacc ctggtgatctgcctcctgtg ggccctctcc 720 ttcatcagca tcacccctgt gtggctgtat gccagactcatccccttccc aggaggtgca 780 gtgggctgcg gcatacgcct gcccaaccca gacactgacctctactggtt caccctgtac 840 cagtttttcc tggcctttgc cctgcctttt gtggtcatcacagccgcata cgtgaggatc 900 ctgcagcgca tgacgtcctc agtggccccc gcctcccagcgcagcatccg gctgcggaca 960 aagagggtga cccgcacagc catcgccatc tgtctggtcttctttgtgtg ctgggcaccc 1020 tactatgtgc tacagctgac ccagttgtcc atcagccgcccgaccctcac ctttgtctac 1080 ttatacaatg cggccatcag cttgggctat gccaacagctgcctcaaccc ctttgtgtac 1140 atcgtgctct gtgagacgtt ccgcaaacgc ttggtcctgtcggtgaagcc tgcagcccag 1200 gggcagcttc gcgctgtcag caacgctcag acggctgacgaggagaggac agaaagcaaa 1260 ggcacctga 1269 2 422 PRT Homo sapiens 2 MetSer Val Gly Ala Met Lys Lys Gly Val Gly Arg Ala Val Gly Leu 1 5 10 15Gly Gly Gly Ser Gly Cys Gln Ala Thr Glu Glu Asp Pro Leu Pro Asp 20 25 30Cys Gly Ala Cys Ala Pro Gly Gln Gly Gly Arg Arg Trp Arg Leu Pro 35 40 45Gln Pro Ala Trp Val Glu Gly Ser Ser Ala Arg Leu Trp Glu Gln Ala 50 55 60Thr Gly Thr Gly Trp Met Asp Leu Glu Ala Ser Leu Leu Pro Thr Gly 65 70 7580 Pro Asn Ala Ser Asn Thr Ser Asp Gly Pro Asp Asn Leu Thr Ser Ala 85 9095 Gly Ser Pro Pro Arg Thr Gly Ser Ile Ser Tyr Ile Asn Ile Ile Met 100105 110 Pro Ser Val Phe Gly Thr Ile Cys Leu Leu Gly Ile Ile Gly Asn Ser115 120 125 Thr Val Ile Phe Ala Val Val Lys Lys Ser Lys Leu His Trp CysAsn 130 135 140 Asn Val Pro Asp Ile Phe Ile Ile Asn Leu Ser Val Val AspLeu Leu 145 150 155 160 Phe Leu Leu Gly Met Pro Phe Met Ile His Gln LeuMet Gly Asn Gly 165 170 175 Val Trp His Phe Gly Glu Thr Met Cys Thr LeuIle Thr Ala Met Asp 180 185 190 Ala Asn Ser Gln Phe Thr Ser Thr Tyr IleLeu Thr Ala Met Ala Ile 195 200 205 Asp Arg Tyr Leu Ala Thr Val His ProIle Ser Ser Thr Lys Phe Arg 210 215 220 Lys Pro Ser Val Ala Thr Leu ValIle Cys Leu Leu Trp Ala Leu Ser 225 230 235 240 Phe Ile Ser Ile Thr ProVal Trp Leu Tyr Ala Arg Leu Ile Pro Phe 245 250 255 Pro Gly Gly Ala ValGly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr 260 265 270 Asp Leu Tyr TrpPhe Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu 275 280 285 Pro Phe ValVal Ile Thr Ala Ala Tyr Val Arg Ile Leu Gln Arg Met 290 295 300 Thr SerSer Val Ala Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr 305 310 315 320Lys Arg Val Thr Arg Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val 325 330335 Cys Trp Ala Pro Tyr Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser 340345 350 Arg Pro Thr Leu Thr Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser Leu355 360 365 Gly Tyr Ala Asn Ser Cys Leu Asn Pro Phe Val Tyr Ile Val LeuCys 370 375 380 Glu Thr Phe Arg Lys Arg Leu Val Leu Ser Val Lys Pro AlaAla Gln 385 390 395 400 Gly Gln Leu Arg Ala Val Ser Asn Ala Gln Thr AlaAsp Glu Glu Arg 405 410 415 Thr Glu Ser Lys Gly Thr 420 3 1214 DNA rat 3gcaggcgacc tgcaccggct gcatggatct gcaaacctcg ttgctgtcca ctggccccaa 60tgccagcaac atctccgatg gccaggataa tctcacattg ccggggtcac ctcctcgcac 120agggagtgtc tcctacatca acatcattat gccttccgtg tttggtacca tctgtctcct 180gggcatcgtg ggaaactcca cggtcatctt tgctgtggtg aagaagtcca agctacactg 240gtgcagcaac gtccccgaca tcttcatcat caacctctct gtggtggatc tgctcttcct 300gctgggcatg cctttcatga tccaccagct catggggaac ggcgtctggc actttgggga 360aaccatgtgc accctcatca cagccatgga cgccaacagt cagttcacta gcacctacat 420cctgactgcc atgaccattg accgctactt ggccaccgtc caccccatct cctccaccaa 480gttccggaag ccctccatgg ccaccctggt gatctgcctc ctgtgggcgc tctccttcat 540cagtatcacc cctgtgtggc tctacgccag gctcattccc ttcccagggg gtgctgtggg 600ctgtggcatc cgcctgccaa acccggacac tgacctctac tggttcactc tgtaccagtt 660tttcctggcc tttgcccttc cgtttgtggt cattaccgcc gcatacgtga aaatactaca 720gcgcatgacg tcttcggtgg ccccagcctc ccaacgcagc atccggcttc ggacaaagag 780ggtgacccgc acggccattg ccatctgtct ggtcttcttt gtgtgctggg caccctacta 840tgtgctgcag ctgacccagc tgtccatcag ccgcccgacc ctcacgtttg tctacttgta 900caacgcggcc atcagcttgg gctatgctaa cagctgcctg aacccctttg tgtacatagt 960gctctgtgag acctttcgaa aacgcttggt gttgtcagtg aagcctgcag cccaggggca 1020gctccgcacg gtcagcaacg ctcagacagc tgatgaggag aggacagaaa gcaaaggcac 1080ctgacaattc cccagtcgcc tccaagtcag gccaccccat caaaccgtgg ggagagatac 1140tgagattaaa cccaaggcta ccctgggaga atgcagaggc tggaggctgg gggcttgtag 1200caaccacatt ccac 1214 4 353 PRT rat 4 Met Asp Leu Gln Thr Ser Leu Leu SerThr Gly Pro Asn Ala Ser Asn 1 5 10 15 Ile Ser Asp Gly Gln Asp Asn LeuThr Leu Pro Gly Ser Pro Pro Arg 20 25 30 Thr Gly Ser Val Ser Tyr Ile AsnIle Ile Met Pro Ser Val Phe Gly 35 40 45 Thr Ile Cys Leu Leu Gly Ile ValGly Asn Ser Thr Val Ile Phe Ala 50 55 60 Val Val Lys Lys Ser Lys Leu HisTrp Cys Ser Asn Val Pro Asp Ile 65 70 75 80 Phe Ile Ile Asn Leu Ser ValVal Asp Leu Leu Phe Leu Leu Gly Met 85 90 95 Pro Phe Met Ile His Gln LeuMet Gly Asn Gly Val Trp His Phe Gly 100 105 110 Glu Thr Met Cys Thr LeuIle Thr Ala Met Asp Ala Asn Ser Gln Phe 115 120 125 Thr Ser Thr Tyr IleLeu Thr Ala Met Thr Ile Asp Arg Tyr Leu Ala 130 135 140 Thr Val His ProIle Ser Ser Thr Lys Phe Arg Lys Pro Ser Met Ala 145 150 155 160 Thr LeuVal Ile Cys Leu Leu Trp Ala Leu Ser Phe Ile Ser Ile Thr 165 170 175 ProVal Trp Leu Tyr Ala Arg Leu Ile Pro Phe Pro Gly Gly Ala Val 180 185 190Gly Cys Gly Ile Arg Leu Pro Asn Pro Asp Thr Asp Leu Tyr Trp Phe 195 200205 Thr Leu Tyr Gln Phe Phe Leu Ala Phe Ala Leu Pro Phe Val Val Ile 210215 220 Thr Ala Ala Tyr Val Lys Ile Leu Gln Arg Met Thr Ser Ser Val Ala225 230 235 240 Pro Ala Ser Gln Arg Ser Ile Arg Leu Arg Thr Lys Arg ValThr Arg 245 250 255 Thr Ala Ile Ala Ile Cys Leu Val Phe Phe Val Cys TrpAla Pro Tyr 260 265 270 Tyr Val Leu Gln Leu Thr Gln Leu Ser Ile Ser ArgPro Thr Leu Thr 275 280 285 Phe Val Tyr Leu Tyr Asn Ala Ala Ile Ser LeuGly Tyr Ala Asn Ser 290 295 300 Cys Leu Asn Pro Phe Val Tyr Ile Val LeuCys Glu Thr Phe Arg Lys 305 310 315 320 Arg Leu Val Leu Ser Val Lys ProAla Ala Gln Gly Gln Leu Arg Thr 325 330 335 Val Ser Asn Ala Gln Thr AlaAsp Glu Glu Arg Thr Glu Ser Lys Gly 340 345 350 Thr 5 26 DNA ArtificialSequence Description of Artificial Sequence primer/probe 5 gggaactccacggtcatctt cgcggt 26 6 26 DNA Artificial Sequence Description ofArtificial Sequence primer/probe 6 tagcggtcaa tggccatggc ggtcag 26 7 45DNA Artificial Sequence Description of Artificial Sequence primer/probe7 ctcctgggca tgcccttcat gatccaccag ctcatgggca atggg 45 8 25 DNAArtificial Sequence Description of Artificial Sequence primer/probe 8cttctaggcc tgtacggaag tgtta 25 9 27 DNA Artificial Sequence Descriptionof Artificial Sequence primer/probe 9 gttgtggttt gtccaaactc atcaatg 2710 37 DNA Artificial Sequence Description of Artificial Sequenceprimer/probe 10 cgcggatcca ttatgtctgc actccgaagg aaatttg 37 11 38 DNAArtificial Sequence Description of Artificial Sequence primer/probe 11cgcgaattct tatgtgaagc gatcagagtt catttttc 38 12 34 DNA ArtificialSequence Description of Artificial Sequence primer/probe 12 gcgggatccgctatggctgg tgattctagg aatg 34 13 29 DNA Artificial Sequence Descriptionof Artificial Sequence primer/probe 13 ccggaattcc cctcacaccg agcccctgg29 14 20 DNA Artificial Sequence Description of Artificial Sequenceprimer/probe 14 tcagctcggt tgtgggagca 20 15 18 DNA Artificial SequenceDescription of Artificial Sequence primer/probe 15 cttggacttc ttcacgac18

What is claimed is:
 1. A process involving competitive binding foridentifying a chemical compound which specifically binds to a ratMelanin-concentrating hormone 1 (MCH1) receptor which comprisescontacting cells expressing on their cell surface the rat MCH1 receptor,with both the chemical compound and a second chemical compound known tobind to the receptor, and separately with only the second chemicalcompound, under conditions suitable for binding of both compounds, anddetecting specific binding of the chemical compound to the rat MCH1receptor, a decrease in the binding of the second chemical compound tothe rat MCH1 receptor in the presence of the chemical compound relativeto the binding of the second chemical compound in the absence of thechemical compound indicating that the chemical compound binds to the ratMCH1 receptor, wherein the cells do not normally express the rat MCH1receptor, the DNA encoding the rat MCH1 receptor has the definedsequence shown in FIG. 4 (SEQ ID NO: 3) and the second chemical compoundis Melanin-concentrating hormone (MCH) or an analog of MCH.
 2. A processinvolving competitive binding for identifying a chemical compound whichspecifically binds to a rat Melanin-concentrating hormone 1 (MCH1)receptor which comprises contacting a membrane preparation from cellsexpressing on their cell surface the rat MCH1 receptor, with both thechemical compound and a second chemical compound known to bind to thereceptor, and separately with only the second chemical compound, underconditions suitable for binding of both compounds, and detectingspecific binding of the chemical compound to the rat MCH1 receptor, adecrease in the binding of the second chemical compound to the rat MCH1receptor in the presence of the chemical compound relative to thebinding of the second chemical compound in the absence of the chemicalcompound indicating that the chemical compound binds to the rat MCH1receptor, wherein the cells do not normally express the rat MCH1receptor, the DNA encoding the rat MCH1 receptor has the definedsequence shown in FIG. 4 (SEQ ID NO: 3) and the second chemical compoundis Melanin-concentrating hormone (MCH) or an analog of MCH.
 3. Theprocess of claim 1 or 2 wherein the cell is an insect cell.
 4. Theprocess of claim 1 or 2, wherein the cell is a manmmalian cell.
 5. Theprocess of claim 4, wherein the cell is nonneuronal in origin.
 6. Theprocess of claim 5, wherein the nonneuronal cell is a COS-7 cell, 293human embryonic kidney cell, a CHO cell, a NIH-3T3 cell, a mouse Y1cell, or a LM(tk-) cell.
 7. A process for preparing a composition whichcomprises admixing a pharmaceutically acceptable carrier and a chemicalcompound identified by the process of claim 1 or
 2. 8. A process fordetermining whether a chemical compound is a rat Melanin-concentratinghormone 1 (MCH1) receptor antagonist which comprises contacting cellstransfected with and expressing DNA encoding the rat MCH1 receptor withthe compound in the presence of a known rat MCH1 receptor agonist, underconditions permitting the activation of the rat MCH1 receptor, anddetecting rat MCH1 receptor activity, wherein a decrease in rat MCH1receptor activity in the presence of both the compound and the knownagonist relative to the activity of the rat MCH1 receptor in thepresence of the known agonist alone indicates that the compound is a ratMCH1 receptor antagonist, wherein the cells do not normally express therat MCH1 receptor, the DNA encoding the rat MCH1 receptor has thedefined sequence shown in FIG. 4 (SEQ ID NO: 3) and the known rat MCH1receptor agonist is Melanin-concentrating hormone (MCH) or an analog ofMCH.
 9. A process for determining whether a chemical compoundspecifically binds to and inhibits activation of a ratMelanin-concentrating hormone 1 (MCH1) receptor, which comprisesseparately contacting cells capable of producing a second messengerresponse and expressing on their cell surface the rat MCH1 receptor,wherein such cells do not normally express the rat MCH1 receptor, withboth the chemical compound and a second chemical compound known toactivate the rat MCH1 receptor, and with only the second chemicalcompound, under conditions suitable for activation of the rat MCH1receptor, and measuring the second messenger response in the presence ofonly the second chemical compound and in the presence of both the secondchemical compound and the chemical compound, a smaller change in thesecond messenger response in the presence of both the chemical compoundand the second chemical compound than in the presence of only the secondchemical compound indicating that the chemical compound inhibitsactivation of the rat MCH1 receptor, wherein the DNA encoding the ratMCH1 receptor has the defined sequence shown in FIG. 4 (SEQ ID NO: 3)and the second chemical compound is Melanin-concentrating hormone (MCH)or an analog of MCH.
 10. A process for preparing a composition whichcomprises admixing a pharmaceutically acceptable carrier and a chemicalcompound identified by the process of claim 8 or
 9. 11. The process ofclaim 9, wherein the second messenger response comprises chloridechannel activation and the change in second messenger response is asmaller increase in the level of inward chloride current in the presenceof both the chemical compound and the second chemical compound than inthe presence of only the second chemical compound.
 12. The process ofclaim 9 or 11, wherein the cell is an insect cell.
 13. The process ofclaim 9 or 11, wherein the cell is a mammalian cell.
 14. The process ofclaim 13, wherein the mammalian cell is nonneuronal in origin.
 15. Theprocess of claim 14, wherein the nonneuronal cell is a COS-7 cell, CHOcell, 293 human embryonic kidney cell, NIH-3T3 cell or LM(tk-) cell.